Evolution of Colistin Resistance in the Klebsiella pneumoniae Complex Follows Multiple Evolutionary Trajectories with Variable Effects on Fitness and Virulence Characteristics
This article has been corrected.
VIEW CORRECTIONABSTRACT
The increasing prevalence of multidrug-resistant Klebsiella pneumoniae has led to a resurgence in the use of colistin as a last-resort drug. Colistin is a cationic antibiotic that selectively acts on Gram-negative bacteria through electrostatic interactions with anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPSs). Colistin resistance in K. pneumoniae is mediated through loss of these phosphate groups, their modification by cationic groups, and by the hydroxylation of acyl groups of lipid A. Here, we study the in vitro evolutionary trajectories toward colistin resistance in four clinical K. pneumoniae complex strains and their impact on fitness and virulence characteristics. Through population sequencing during in vitro evolution, we found that colistin resistance develops through a combination of single nucleotide polymorphisms, insertions and deletions, and the integration of insertion sequence elements, affecting genes associated with LPS biosynthesis and modification and capsule structures. Colistin resistance decreased the maximum growth rate of one K. pneumoniae sensu stricto strain, but not those of the other three K. pneumoniae complex strains. Colistin-resistant strains had lipid A modified through hydroxylation, palmitoylation, and l-Ara4N addition. K. pneumoniae sensu stricto strains exhibited cross-resistance to LL-37, in contrast to the Klebsiella variicola subsp. variicola strain. Virulence, as determined in a Caenorhabditis elegans survival assay, was increased in two colistin-resistant strains. Our study suggests that nosocomial K. pneumoniae complex strains can rapidly develop colistin resistance through diverse evolutionary trajectories upon exposure to colistin. This effectively shortens the life span of this last-resort antibiotic for the treatment of infections with multidrug-resistant Klebsiella.
INTRODUCTION
Klebsiella pneumoniae is a Gram-negative opportunistic pathogen and a leading cause of hospital-associated infections such as pneumonia, surgical site infections, and urinary tract infections. K. pneumoniae may also asymptomatically colonize the skin, upper respiratory tract, and digestive tract of healthy individuals (1, 2). The K. pneumoniae complex is genetically diverse, with different phylogroups within the complex corresponding to different species and subspecies, each occupying specific niches (1, 2). The K. pneumoniae sensu strico and Klebsiella quasipneumoniae phylogroups are associated with human intestinal carriage, while the Klebsiella variicola phylogroup is associated with plants and bovines (1, 3). Of all strains isolated from human infections and typed as K. pneumoniae, the majority are K. pneumoniae sensu stricto, but K. variicola and K. quasipneumoniae have also been found to cause infections in patients and are frequently misidentified as K. pneumoniae (4, 5). Although infections with strains from the K. variicola phylogroup are relatively rare, they have been associated with the highest mortality rate within the K. pneumoniae complex (3).
In recent years, K. pneumoniae complex strains have rapidly emerged as multidrug-resistant pathogens through acquisition of resistance to third-generation cephalosporins, fluoroquinolones, and aminoglycosides, and they have increasingly become resistant to carbapenems through the acquisition of carbapenemases (6–9). The increasing prevalence of multidrug resistance within the K. pneumoniae complex and the lack of development of novel antibiotic classes effective against Gram-negative bacteria have limited the available therapeutic options against multidrug-resistant K. pneumoniae complex strains. These limitations have prompted the resurgence in the use of the antibiotic colistin in treatment of infections by K. pneumoniae complex strains (10–12). After its introduction into clinical practice in the 1950s, colistin fell into disuse in human medicine in the 1970s because of the neuro- and nephrotoxic side effects associated with its use and the development of safer classes of antibiotics. Due to the emergence of multidrug-resistant Gram-negative opportunistic pathogens, like K. pneumoniae, it has recently regained clinical relevance as a last-line antibiotic (13).
Colistin (polymyxin E) is a cationic, amphipathic molecule composed of a fatty acid chain linked to a nonribosomally synthesized decapeptide (14, 15). The mechanism of action of colistin relies on the selective presence of the negatively charged lipopolysaccharides (LPS) in the membranes of Gram-negative bacteria. The negative charges of LPS are carried by the anionic phosphate groups of the lipid A moiety of LPS, which enables colistin to bind through electrostatic interactions (14). Insertion of colistin into the outer membrane leads to membrane permeabilization. The subsequent destabilization of the cytoplasmic membrane, where LPS is present after synthesis in the cytoplasm while awaiting transport to the outer membrane, ultimately leads to cell death (14, 16, 17).
The increased use of colistin to treat infections with multidrug-resistant Gram-negative bacteria, especially in low- and middle-income countries (12), and the use of colistin in livestock farming, either therapeutically to treat enteric infections or as a growth promoter (18), have led to a rise in colistin resistance in K. pneumoniae from clinical, veterinary, and environmental sources (9, 18, 19). Colistin resistance in K. pneumoniae complex strains is mostly mediated through decoration of lipid A with cationic groups to counteract the electrostatic interactions between colistin and lipid A (14). These modifications can be the result of point mutations and insertions/deletions (indels) in chromosomally located genes (including phoPQ, pmrAB, and crrAB) resulting in amino acid substitutions, insertions, and deletions in the proteins encoded by these genes (20–23). In addition, the acquisition of mobile genetic elements carrying a member of the mcr gene family may also lead to lipid A modification (23, 24). In K. pneumoniae, the inactivation of mgrB encoding a negative regulator of the two-component regulatory system PhoPQ, through the insertion of an insertion sequence (IS) element or a mutation leading to the formation of a premature stop codon, is a particularly frequently observed colistin resistance mechanism (25–29). Other mechanisms of colistin resistance in K. pneumoniae include the upregulated expression of efflux pumps (30, 31), changes in LPS production (20, 32), and the overproduction of capsular polysaccharides (33, 34).
Upon infection, the innate immune system will attempt to neutralize invading bacteria. The cellular components of the innate immune system can detect Gram-negative bacteria through the presence of LPS (35). Activated immune cells can kill bacteria and will attempt to kill them by unleashing bactericidal components, including the antimicrobial peptide LL-37. Similar to colistin, LL-37 relies on electrostatic interactions with LPS for its mechanism of action (36). Modifications to LPS may influence the efficacy of bactericidal components and may thus result in altered virulence by reducing the effectiveness of these components (35–37). Modifications capable of affecting the efficiency of the immune system include neutralization of the anionic charges carried by lipid A and changes in acylation of lipid A (35, 38, 39). These changes are mediated through the PhoPQ and PmrAB two-component regulatory systems. Notably, colistin resistance is mediated through the same modifications and two-component regulatory systems. The development of colistin resistance may thus also affect virulence characteristics.
To better understand the mechanisms and consequences of colistin resistance in K. pneumoniae complex strains, we determined the evolutionary trajectories of three K. pneumoniae sensu stricto strains and one K. variicola subsp. variicola strain toward colistin resistance in an in vitro evolution experiment and determined how colistin resistance impacted fitness, LPS modifications, and virulence characteristics.
RESULTS
The colistin-susceptible K. pneumoniae complex strains have a diverse genetic background.
The four clinical isolates used in this study were obtained from pus, fecal, or urine samples through routine diagnostic procedures in September 2013. All four strains were initially typed as K. pneumoniae sensu stricto through routine diagnostic procedures using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The susceptibility to colistin of these strains, previously determined in routine diagnostic procedures, was confirmed through antibiotic susceptibility testing using broth microdilution (Fig. 1A).
FIG 1
The sequenced genomes of the colistin-susceptible strains were screened for acquired antibiotic resistance genes through ResFinder 3.1 (Fig. 1B). None of the nosocomial strains was determined to carry one of the mcr genes. Between two and five acquired antibiotic resistance genes were observed in the genome assemblies, encoding resistance to β-lactams, quinolones, and fosfomycin.
To accurately identify the phylogenetic position of these nosocomial strains within the K. pneumoniae complex, a phylogenetic tree was generated based on the Illumina/Oxford Nanopore hybrid genome assemblies of the colistin-susceptible strains and 37 publicly available genomes covering all phylogroups in the K. pneumoniae complex (2). Based on a 1.3-Mbp core genome alignment, the phylogenetic tree showed that strains KP209, KP040, and KP257 clustered in the K. pneumoniae sensu stricto (KpI) phylogroup (Fig. 1C). Strain KV402 clustered in the K. variicola subsp. variicola (KpIII) phylogroup, even though it had been typed as K. pneumoniae sensu stricto through MALDI-TOF MS during initial routine diagnostic procedures.
Colistin resistance emerges through multiple evolutionary trajectories in the K. pneumoniae complex.
To understand the evolutionary trajectories through which the K. pneumoniae complex strains evolved resistance toward colistin, we deep sequenced each overnight culture during growth in increasing concentrations of colistin (see Table S2 in the supplemental material) and identified single nucleotide polymorphisms (SNPs), indels, and excision/integration events of IS elements.
We observed the rapid emergence and fixation of several mutations (Fig. 2) in the presence of colistin. In three populations (KP209, KP257, and KV402), these mutations occurred in the genes encoding the PhoPQ two-component regulatory system after 1 day of culturing (see Table S3 in the supplemental material). In the KP040 population, we observed the integration of an IS5 element (see Table S4 and Fig. S2 in the supplemental material) in the promoter region of both the crrAB operon and the divergently transcribed crrC gene. In addition, an intergenic SNP (located in promoter regions of ecpR or phnC) in KP040 became fixed in the population on the first day of culturing. Both EcpR and PhnC had not previously been associated with colistin resistance. Although other mutations in other locations also occurred during the first day of culturing, these mutations failed to become fixed in the population and were either lost on subsequent days or did not change in abundance over time.
FIG 2
On subsequent days of the in vitro evolution experiment, novel mutations in the populations were associated with additional increases in the MIC of colistin. New SNPs that were fixed in the populations were observed in phoQ (KP209 on day 5 and KV402 on day 6) and pmrB (KP209 on day 4). In KP257, an SNP in lptD was first observed on day 3 and was then fixed in the population. The lptD gene encodes a barrel-shaped transporter that transports LPS onto the outer leaflet of the outer membrane (40). Mutations in genes located in the capsule synthesis locus (K-locus) were also detected. In KV402, a 13-bp deletion was observed in wcaJ from day 3 onwards, leading to a premature stop codon. In KP040, a new insertion of IS102, inactivating wzc, was observed from day 4. In addition, a 12-bp insertion in the gene encoding the Rho transcription termination factor was observed in KP040. We did not observe any mutations in the mgrB gene in these in vitro evolution experiments.
K. pneumoniae can rapidly develop colistin resistance without loss of fitness.
To enable a further characterization of the impact of the evolution of colistin resistance on fitness and virulence characteristics, we isolated a single random colony on nonselective medium from each day of the in vitro evolution experiments. The genome sequences of the axenic strains of the last day of the in vitro evolution experiments were determined by Illumina sequencing. SNPs, indels, and IS element insertions were identified in these strains in comparison with the colistin-susceptible parental strain. After combining these data with the population sequencing data described above, we determined the presence of these mutations in the axenic strains isolated after each day of the in vitro evolution experiment by targeted PCRs and Sanger sequencing of the amplicons. We were thus able to correlate the occurrence of mutations with increases in the MIC of colistin (determined in cation-adjusted Mueller-Hinton II broth [MHCAB]) in each strain.
All four strains developed levels of resistance to colistin above the breakpoint value (2 μg/ml) after one overnight incubation of the colistin-susceptible strain (MIC of ≤2 μg/ml) in the presence of the antibiotic (Fig. 2). The initial mutations in phoPQ were associated with an increase in the MIC in strains KP209 (32 μg/ml), KP257 (128 μg/ml), and KV402 (32 μg/ml) (Fig. 2). The integration of the IS5 element in the promoter region of crrAB and crrC and the appearance of an intergenic SNP between ecpR and phnC also occur simultaneously with an increase in the MIC of colistin (4 μg/ml). The additional SNP in phoQ in KP209 was not associated with an increase in the MIC of colistin (256 μg/ml). Integration of IS102 in wzc of the K-locus, as well as the 12-bp insertion in the gene encoding the transcription termination factor Rho, was associated with an additional increase in the MIC of colistin (128 μg/ml) in strain KP040. The SNP in lptD in strain KP257 did not lead to a meaningful increase in the MIC of colistin (256 μg/ml). The culture isolated from the last day of the KV402 in vitro evolution experiments had an SNP in yciM (see Table S5 in the supplemental material), encoding a negative regulator of LPS biosynthesis (20), but this did not contribute to a further reduced susceptibility to colistin. Because of the random nature of picking single isolates from their populations, some mutations identified by population sequencing were not recapitulated in the axenic strains and vice versa (Fig. 2; Table S5).
The measurement of the maximum growth rate as a proxy for general fitness of the axenic strains isolated on the different days of the in vitro evolution experiment showed that the increase in MIC of colistin to values above 2 μg/ml after one overnight incubation did not negatively affect the maximum growth rate for strains KP209, KP040, and KV402. Only the initial increase in the MIC of colistin in strain KP257 had a negative impact on the maximum growth rate, decreasing the maximum growth rate by 37% (Fig. 3). Over time, the maximum growth rates of strains KP209 and KV402 decreased 13.4% and 9.5%, respectively, compared to the maximum growth rate of the colistin-susceptible strain. In strain KP040, an increase of 10.0% in maximum growth rate was observed during the course of the in vitro evolution experiment.
FIG 3
Colistin-resistant K. pneumoniae complex strains have lipid A that is modified through hydroxylation, palmitoylation, and addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N).
To determine the modifications to lipid A in the colistin-resistant strains, we performed MALDI-TOF analysis on lipid A isolated from the colistin-susceptible strain and the axenic strain of the last day of the in vitro evolution experiments. The MALDI-TOF spectra of lipid A isolated from colistin-susceptible strains (Fig. 4A) showed a dominant peak from hexa-acylated lipid A (mass/charge ratio [m/z] of 1,824), corresponding to two glucosamines, two phosphates, and four 3-OH-C14 and two C14 acyl chains (41). Additional minor peaks in the MALDI-TOF spectrum of the susceptible strains could be observed at an m/z of 1,840, corresponding to the hydroxylation (m/z of 16) of one of the C14 acyl groups of hexa-acylated lipid A (m/z of 1,824), and at an m/z of 2,063 (in KP209 and KP257), corresponding to a hepta-acylated lipid A, with an additional acylation of lipid A (m/z of 1,824) with a palmitoyl group (m/z of 239).
FIG 4
All the MALDI-TOF spectra of lipid A isolated from colistin-resistant strains show additional peaks (Fig. 4B), indicating the modification of their lipid A. In the spectra of colistin-resistant KP209 and KV402, a lipid A m/z of 1,955 was observed, indicating addition of l-Ara4N (m/z of 131) to the hexa-acylated lipid A m/z of 1,824. In colistin-resistant KV402 lipid A, an m/z of 1,850 was observed, consistent with a hexa-acylated lipid A m/z of 1,824 with one C16 acyl chain (Fig. 4C). The peak at an m/z of 1,866 in the MALDI-TOF spectra of colistin-resistant KP040 and KP257 was consistent with hydroxylation of a lipid A m/z of 1,850.
Development of colistin resistance is associated with increased LL-37 resistance and virulence in a C. elegans survival model.
To determine the impact of colistin resistance on virulence characteristics of the K. pneumoniae complex strains, we first determined the susceptibility of the strains to the human cathelicidin antimicrobial peptide LL-37. We observed that three of the four colistin-resistant strains (KP209, KP040, and KP257) showed a decreased susceptibility to killing by LL-37 compared to their colistin-susceptible parental strains (Fig. 5). In contrast, development of colistin resistance in strain KV402 did not affect susceptibility to LL-37.
FIG 5
To investigate the possible consequences of colistin resistance on virulence, we exposed the nematode Caenorhabditis elegans strain CF512 to the colistin-susceptible and -resistant strain pairs. C. elegans had a decreased life span on a lawn of colistin-resistant KP209 (Fig. 6) and KP040, compared to their colistin-susceptible strains. Survival of C. elegans was not affected by growth on colistin-resistant strains derived from KP257 and KV402, compared to the colistin-susceptible parental strains.
FIG 6
DISCUSSION
Colistin plays a pivotal role in public health due to its last-resort status for treatment of infections with multidrug-resistant Gram-negative bacteria. The increasing number of reports of K. pneumoniae strains that have acquired resistance to multiple antibiotics, including colistin, is thus a cause for increasing concern (7–9, 42). In this study, we aimed to study the potential to evolve colistin resistance in clinical K. pneumoniae isolates. Due to the difficulties in generating targeted mutants in these multidrug-resistant clinical isolates, we were limited to in vitro evolution experiments to identify mutations associated with colistin resistance. We observed the swift development of colistin resistance through diverse evolutionary trajectories. Development of colistin resistance had no, or only a minor, impact on maximum growth rate in three out of four in vitro evolution experiments performed here. This suggests that colistin may rapidly lose its effectiveness in the treatment of infections caused by multidrug-resistant K. pneumoniae complex strains as fitness costs associated with colistin resistance seem limited.
We observe that mutations associated with an increase in the MIC of colistin seem confined to genes from functional groups involved in the synthesis and modification of LPS and the synthesis of capsular polysaccharides, which are both important surface-associated structures. In the genes encoding the PhoPQ two-component regulatory system, which have a role in regulating modifications of LPS and contribute to colistin resistance in Enterobacteriaceae (21, 43), we found variations in both PhoP (a D191N substitution) and PhoQ (a G385S substitution and a 12-bp deletion). The G385S PhoQ substitution has previously been described in a colistin-resistant clinical K. pneumoniae strain (44). We also found that a novel integration of an IS5 element in the promoter region associated with the genes encoding CrrAB and CrrC coincides with an increase in the MIC of colistin. The IS5 element can influence the transcriptional activity of the genes located near its integration site (45). The activity of PmrAB may be influenced by CrrAB through CrrC (22, 46). In line with previous observations, in which insertions of IS elements were associated with resistance to colistin, we hypothesize that the insertion of IS5 may lead to increased expression of CrrAB and/or CrrC and thus cause colistin resistance (28).
We observed that the inactivation of wzc of the K-locus by the IS102 element coincides with an increase in the MIC of colistin. In Escherichia coli, Wzc is involved in the synthesis and export of extracellular polysaccharides containing colanic acid (47), but also the phosphorylation of other endogenous proteins (48). Wzc has previously been hypothesized to be involved in colistin resistance in E. coli, and it may act similarly in K. pneumoniae (48–50). The loss of Wzc may potentially cause colistin resistance through two mechanisms. A reduction in the export of colanic acid units (the building blocks of K. pneumoniae capsule) can lead to the accumulation of colanic acid metabolic intermediates, including UDP-glucuronic acid. This accumulation has been hypothesized to lead to an increased flux toward biosynthesis of UDP-l-Ara4N, resulting in the modification of lipid A with l-Ara4N (51). Alternatively, the absence or reduction of negatively charged colanic acid residues on the cell surface could lower local concentrations of positively charged colistin molecules, thereby reducing damage to the outer membrane (51). Further studies are needed to fully characterize the interplay between the Klebsiella capsule and colistin resistance. In addition to the inactivation of wzc, we observed a 12-bp insertion in the highly conserved rho gene, encoding the transcription termination factor Rho. Rho has not been previously linked to colistin resistance, but mutations in rho may have pleiotropic effects on transcription (52), which could influence the expression of genes involved in, or may compensate for fitness costs caused by, colistin resistance.
Notably, we did not find any alterations in mgrB, which is an otherwise important mechanism through which colistin resistance may occur in nosocomial K. pneumoniae complex strains (20, 25–27, 53). Nevertheless colistin-resistant clinical K. pneumoniae isolates without mutations in mgrB are also frequently encountered (44, 46, 54–57). We can only speculate on the reasons for the absence of mgrB mutations in our in vitro evolution experiments. The relatively short duration of this experiment performed with a limited number of strains likely implies that we have not covered all potential colistin resistance mechanisms in K. pneumoniae. Due to these limitations and the lack of replicate experiments, we cannot make any conclusions on the repeatability or the need for a specific order in these mutational pathways.
The impact of developing colistin resistance through the observed mutations might extend past the inability to treat the infection through antibiotic therapy, as we show in this study that modifications to lipid A may reduce the susceptibility to antimicrobial peptides and increase virulence. However, the mechanisms behind the differential effects on virulence of colistin resistance in the K. pneumoniae complex are not fully understood and are deserving of further study. A single K. variicola isolate was included in this study. While K. variicola can cause life-threatening infections in immunocompromised individuals (5), it remains currently understudied. Additional studies of the mechanisms of colistin resistance and their impact on fitness and virulence may be warranted in this species.
The emergence and spread of colistin resistance could complicate future treatments of infections caused by multidrug-resistant Gram-negative bacteria. Our study indicates that in the K. pneumoniae complex, multiple evolutionary trajectories toward colistin resistance exist, without negatively impacting fitness or virulence characteristics. Our data highlight the remarkable adaptive abilities of strains in the K. pneumoniae complex, which makes them nosocomial pathogens of considerable importance. Future studies may lead to the development of novel therapeutics to specifically target colistin resistance mechanisms, which may be essential to lengthen the clinical life span of colistin as a last-resort drug in treatment of K. pneumoniae infections.
MATERIALS AND METHODS
Ethical statement.
The colistin-susceptible K. pneumoniae complex strains used in this study were isolated as part of routine diagnostic procedures, which did not require consent or ethical approval by an institutional review board.
Bacterial strains, growth conditions, and chemicals.
The colistin-susceptible KP209, KP040, KP257, and KV402 strains were retrospectively obtained from the diagnostic laboratory of the University Medical Center Utrecht, Utrecht, The Netherlands. In initial routine diagnostic procedures, they were identified as K. pneumoniae sensu stricto by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis on a Bruker microflex system (Leiderdorp, The Netherlands). Colistin susceptibility testing of the clinical isolates was initially performed on a BD Phoenix automated identification and susceptibility testing system (Becton Dickinson, Vianen, The Netherlands). All strains were grown either in lysogeny broth (LB; Oxoid, Landsmeer, The Netherlands) with agitation at 300 rpm or on LB agar at 37°C, unless otherwise specified. Colistin sulfate was obtained from Duchefa Biochemie (Haarlem, The Netherlands).
Determination of MIC of colistin.
MICs of colistin were determined as described previously (58) in line with the recommendations from the joint Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing (EUCAST) Polymyxin Breakpoints Working Group (https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/General_documents/Recommendations_for_MIC_determination_of_colistin_March_2016.pdf). In short, colistin susceptibility testing was performed using BBL cation-adjusted Mueller-Hinton II broth (MHCAB; Becton Dickinson), untreated Nunc 96-well round bottom polystyrene plates (Thermo Fisher Scientific, Landsmeer, The Netherlands), and Breathe-Easy sealing membranes (Sigma-Aldrich, Zwijndrecht, The Netherlands). The MIC was observed after stationary overnight growth at 37°C and was determined to be the lowest concentration at which no visible growth was observed. The breakpoint value for colistin resistance of a MIC of >2 μg/ml was obtained from EUCAST (http://www.eucast.org/clinical_breakpoints/).
In vitro evolution of colistin resistance.
The nosocomial, colistin-susceptible K. pneumoniae strains were evolved toward colistin resistance by culturing in increasing colistin concentrations over a period of 5 to 7 days. As we used LB as the medium for the in vitro evolution, we first determined the colistin MICs in this medium (as outlined above) and subsequently grew each strain in 1 ml LB with initial colistin concentrations of 1× and 2× the MIC. After overnight growth, 1 μl of the cultures with the highest concentration of colistin that had visible growth was used to propagate a fresh culture by inoculating 1 ml of fresh LB, supplemented with the same concentration or twice the concentration of colistin in which growth was observed in the previous day’s culture (see Fig. S1 in the supplemental material). This process was repeated for 5 to 7 days. Each overnight culture was stored at −80°C in 20% glycerol.
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 2.0 fluorometer and the Qubit double-stranded DNA (dsDNA) broad-range assay kit (Life Technologies, Bleiswijk, The Netherlands).
Illumina sequence libraries of genomic DNA were prepared using the Nextera XT kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions and sequenced on an Illumina MiSeq system with a 500-cycle (2 × 250 bp) MiSeq v2 reagent kit (Illumina). MinION library preparation for barcoded two-dimensional (2D) long-read sequencing was performed using the SQK-LSK208 kit (Oxford Nanopore Technologies, Oxford, United Kingdom), according to the manufacturer’s instructions, with G-tube (Covaris, Woburn, MA, USA) shearing of chromosomal DNA for 2 × 120 s at 1,500 g. The libraries were sequenced on a MinION sequencer (Oxford Nanopore Technologies) through a SpotON Flow Cell Mk I (R9.4; Oxford Nanopore Technologies).
Genome assembly and annotation.
The quality of the Illumina sequencing data was assessed using FastQC v0.11.5 (https://github.com/s-andrews/FastQC). Illumina 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. MinION reads in the FastQ format were extracted from Metrichor base-called FAST5 files using Poretools (59). De novo hybrid genome assembly of the colistin-susceptible strains was performed with Illumina and Oxford Nanopore data as described previously (60). Genome annotation was performed using Prokka (61).
Phylogenetic analysis, MLST typing, and identification of antibiotic resistance genes.
To generate a core genome phylogeny, Illumina/Oxford Nanopore hybrid genome assemblies were aligned using ParSNP v1.2 (37) with 37 publicly available Klebsiella pneumoniae complex genomes that cover all phylogroups of the K. pneumoniae complex (2). To include the genome of Klebsiella africanensis strain 38679, we assembled the genome from raw reads, by processing the raw sequence reads using Nesoni with standard settings, except for minimum read length (75 nucleotides), and subsequent assembly by SPAdes with kmers 21, 33, 55, and 77 and the “careful” options turned on.
Figtree was used to visualize and midpoint root the phylogenetic tree (http://tree.bio.ed.ac.uk/). multilocus sequence type (MLST) typing was performed using the mlst package v2.10 (https://github.com/tseemann/mlst). Genome assemblies of colistin-susceptible strains were assessed for antibiotic resistance genes by ResFinder 3.1 through standard settings (62).
Determination of SNPs and indels between axenic colistin-susceptible and colistin-resistant strain pairs.
Read mapping of Nesoni-filtered reads of evolved strains to the genomes of the isogenic colistin-susceptible parental strains was performed using Bowtie2 (63). SNP and indel calling was performed using SAMtools 0.1.18 using the following settings: Qscore of ≥50, mapping quality of ≥30, a mapping depth of ≥10 reads, a consensus of ≥75% to support a call, and ≥1 read in each direction supporting a mutation, as previously described (64). To correct for potential assembly errors, we also performed the SNP and indel calling procedure by mapping the reads of the reference isolates against their own assemblies. SNPs and indels found in the reference-versus-reference comparison were ignored in query-versus-reference comparisons. Synonymous mutations were excluded from further analyses. SNPs and indels were manually linked to genes in the assembly.
Determination of location of IS elements in genomes.
To determine which IS elements were present in the genomes of colistin-susceptible strains, we analyzed the Illumina/Oxford Nanopore hybrid genome assemblies using ISfinder (65). Per genome, the IS elements with an E value of <1e−50 were selected for further study. If multiple distinct IS elements were called at the same position, the element with the highest sequence identity was selected to represent that position.
To detect changes in the position of the identified IS elements, we analyzed the genomic assemblies of the isogenic colistin-susceptible and colistin-resistant strain pairs through ISMapper (66). To maximize the ability of ISMapper to detect IS elements in our sequencing data, the obtained nucleotide sequences of the IS elements in the genome were used as input, and the –cutoff flag of ISMapper was set to 1, while other settings remained unchanged. The results were inspected for IS elements that had different positions between the colistin-susceptible and colistin-resistant strains. Insertion of IS elements was confirmed through PCRs, using DreamTaq green PCR master mix (Thermo Fisher Scientific) and primers spanning the IS insertion site (see Table S1 in the supplemental material) and subsequent Sanger sequencing of the PCR product by Macrogen (Amsterdam, The Netherlands).
SNP and indel calling in evolving populations.
To track the genomic changes within the growing cultures under the selective pressure of increasing colistin concentrations, genomic DNA was isolated from the 5 to 7 overnight cultures of each in vitro evolution experiment and sequenced on the Illumina MiSeq platform as described above. SNPs and indels were called as before, with each call supported by at least 25% of reads. Once identified in one or more populations, the abundances of the specific SNPs and indels were then quantified manually for all individual populations of the in vitro evolution experiment. Mutations called within 150 bp of a contig end were filtered out, as previously recommended (67). Identified SNPs and indels were manually linked to genes in the genome assembly and inspected for synonymous versus nonsynonymous mutations. Noncoding mutations were included in subsequent analyses, while synonymous mutations were excluded.
Determination of growth rate.
To determine the maximum specific growth rate, a Bioscreen C instrument (Oy Growth Curves AB, Helsinki, Finland) was used. Overnight cultures were used to inoculate 200 μl fresh LB medium at 1:1,000. Incubation was set at 37°C with continuous shaking. Growth was observed by measuring the absorbance at 600 nm every 7.5 min. Each experiment was performed in triplicate.
MALDI-TOF analysis of lipid A structures.
Isolation of lipid A molecules and subsequent analysis by negative-ion matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed as previously described (29, 41, 68). Briefly, K. pneumoniae strains were grown in LB (Oxoid), and the lipid A was purified from stationary cultures using the ammonium hydroxide–isobutyric acid isolation method described earlier (69). Mass spectrometry analysis were performed on a Bruker autoflex speed TOF/TOF mass spectrometer in negative reflective mode with delayed extraction using as the 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 analysis. Further calibration for lipid A analysis was performed externally using lipid A extracted from Escherichia coli strain MG1655 grown in LB medium at 37°C.
LL-37 survival assay.
In order to test the susceptibility of the K. pneumoniae strains to LL-37, we adapted previously described protocols (70). An overnight broth culture was diluted to a concentration of 2.5 × 106 CFU/ml in 25% LB and incubated with or without the addition of 50 μg/ml LL-37 (AnaSpec, Inc., Fremont, CA, USA) for 90 min at 37°C with agitation at 300 rpm in sterile round-bottom 96-well plates (Greiner Bio-One, Alphen aan den Rijn, The Netherlands). After incubation, samples were serially diluted in phosphate-buffered saline (PBS) and plated on LB agar plates. CFU were counted after overnight incubation at 37°C.
Caenorhabditis elegans virulence assays.
Caenorhabditis elegans strain CF512 [rrf-3(b26) II; fem-1(hc17) IV], which has a temperature-sensitive reproduction defect, was obtained from the Caenorhabditis Genetics Center at the University of Minnesota, Twin Cities (https://cbs.umn.edu/cgc/home). CF512 nematodes were maintained at 20°C on nematode growth medium (NGM) agar plates seeded with E. coli OP50 (71) and placed on fresh plates at least once per week. For seeding of NGM plates, mid-exponential-phase cultures were used. After reaching mid-exponential phase, the cells were washed with PBS, and 1 × 106 CFU were spread on NGM plates, after which the bacterial lawns were grown overnight at 37°C.
To quantify bacterial virulence, C. elegans CF512 life span assays were performed with synchronized nematodes according to a previously described protocol (72). For synchronization, nematodes and eggs were collected from an NGM plate in ice-cold filter-sterilized M9 medium and washed by spinning at 1,500 × g for 30 s (73). Nematodes were destructed by vigorous vortexing in hypochlorite solution (25 mM NaOH, 1.28% sodium hypochlorite) for 2 min, after which the reaction was stopped by the addition of M9 medium. Eggs were allowed to hatch on NGM plates seeded with E. coli OP50 for 6 to 8 h at 20°C, after which they were placed at 25°C to avoid progeny. After 48 h, L3 to L4 nematodes were placed on NGM plates (n = 40 per plate) seeded with bacterial strains. Plates were scored for live nematodes. Nematodes were considered dead when they did not show spontaneous movement or a response to external stimuli.
Statistical analysis.
Statistical analyses were performed using parametric one-way analysis of variance (ANOVA) with a Dunnett’s test for multiple comparisons (for the determination of maximum growth rates). The nonparametric Mann-Whitney test was used for the LL-37 survival assay, and the Mantel-Cox log rank test was used for the C. elegans assays. Statistical significance was defined as a P value of <0.05 for all tests. Statistical analyses were performed using GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA).
Data availability.
Sequence data from both the Illumina short-read sequencing and the Oxford Nanopore long-read sequencing have been deposited in the European Nucleotide Archive under accession no. PRJEB29521.
ACKNOWLEDGMENTS
We thank the Utrecht Sequence Facility and Ivo Renkens for their expertise in MinION Oxford Nanopore sequencing, Lidewij W. Rümke for the contribution of clinical metadata of the used nosocomial isolates, Inge The for advice on C. elegans assays, and Evelien T. M. Berends for helpful discussions.
W.v.S. was funded by the Netherlands Organisation for Scientific Research through a Vidi grant (grant no. 917.13.357) and a Royal Society Wolfson Research Merit Award (grant no. WM160092). Work in the laboratory of J.A.B. was supported by the Biotechnology and Biological Sciences Research Council BBSRC, (grant no. BB/N00700X/1, BB/P020194/1, and BB/P006078/1) and a Queen’s University Belfast start-up grant. S.H.M.R. was funded by an ERC Starting grant (grant no. 639209-ComBact). The funders had no role in study design, data collection and interpretation, the decision to submit the work for publication, or manuscript preparation.
The authors have declared that no competing interests exist.
A.B.J., D.J.D., and G.M. performed experiments and analyzed data. A.B.J. and M.R.C.R. performed bioinformatic analyses. S.H.M.R., J.A.B., and W.v.S. designed experiments. A.B.J., M.J.M.B., S.H.M.R., R.J.L.W., J.A.B., and W.v.S. wrote the manuscript. All authors reviewed and approved the manuscript prior to submission.
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Antimicrobial Agents and Chemotherapy
Volume 65 • Number 1 • 16 December 2020
eLocator: 10.1128/aac.01958-20
PubMed: 33139278
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Received: 4 October 2020
Accepted: 25 October 2020
Published online: 16 December 2020
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Evolution of Colistin Resistance in the Klebsiella pneumoniae Complex Follows Multiple Evolutionary Trajectories with Variable Effects on Fitness and Virulence Characteristics. Antimicrob Agents Chemother
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