Abstract
Neisseria commensals are an indisputable source of resistance for their pathogenic relatives; however, the evolutionary paths commensal species take to reduced susceptibility in this genus have been relatively underexplored. Here, we leverage in vitro selection as a powerful screen to identify the genetic adaptations that produce azithromycin resistance (≤ 2 μg/mL) in the Neisseria commensal, N. elongata. Across multiple lineages (n=7/16), we find mutations encoding resistance converge on the gene encoding the 50S ribosomal L34 protein (rpmH) and the intergenic region proximal to the 30S ribosomal S3 protein (rpsC) through duplication events. Importantly, one of the laboratory evolved mutations in rpmH is identical, and two nearly identical, to those recently reported to confer high-level resistance to azithromycin in N. gonorrhoeae. Transformations into the ancestral N. elongata lineage confirmed the causality of both rpmH and rpsC mutations. Though most lineages inheriting duplications suffered in vitro fitness costs, one variant showed no growth defect, suggesting the possibility that it may be sustained in natural populations. Finally, we assessed the potential of horizontal transfer of derived resistance mutations into multiple strains of N. gonorrhoeae. Though we were unable to transform N. gonorrhoeae in this case, studies like this will be critical for predicting commensal alleles that are at risk of rapid dissemination into pathogen populations.
Importance Commensal bacterial populations have been increasingly recognized for their importance as sources of resistance for pathogens, however the collection of antimicrobial resistance (AMR) mechanisms within these communities are often understudied. The risk of reduced antibiotic susceptibility as a result of horizontal gene transfer (HGT) is amplified in highly recombinogenic genera, such as the Neisseria. Indeed, there have been multiple documented cases of macrolide and beta-lactam resistance acquisition in the pathogen N. gonorrhoeae from close commensal relatives. This work uncovers multiple novel azithromycin resistance-conferring mutations in a Neisseria commensal through experimental evolution, investigates their fitness impacts, and explores the possibility of transfer to N. gonorrhoeae. Ultimately these types of studies will illuminate those resistance mutations that may rapidly be acquired across species boundaries.
Introduction
Commensal bacterial populations have been increasingly recognized for their importance as sources of adaptive genetic variation for pathogens through horizontal gene transfer (HGT). This threat of rapid evolution as a result of DNA donation is especially amplified in highly recombinogenic genera, such as the Neisseria. Members of this genus readily donate DNA to one another through pilus-mediated Neisseria-specific DNA uptake and homologous recombination (1-3). Observations of genetic mosaicism, whereby loci within a particular lineage have been acquired from another species, are common (4-11) and occur genome-wide (12, 13). This promiscuous allelic exchange has been documented to have facilitated rapid adaptive evolution of important phenotypic characteristics such as antimicrobial resistance (6, 8-10) and body-site colonization niche shifts (11); and overall, recently incorporated mosaic sequences show signatures consistent with positive selection (12), suggesting that intragenus recombination is an important source of beneficial genetic variation.
The genus Neisseria is comprised of several Gram-negative species that typically colonize the mucosa of humans and animals. Most human-associated Neisseria inhabit the naso- and oropharynx, and are carried harmlessly as commensals in 10-15% of healthy human adults and children (14, 15). Only one species, N. gonorrhoeae (Ngo), is an obligate pathogen which also colonizes the urogenital tract and rectum, and causes the sexually transmitted infection gonorrhea (16). In recent years it has become increasingly clear that Neisseria commensals serve as important reservoirs of adaptive genetic variation for Ngo through HGT (6, 8, 9, 12, 17); however, most of the non-pathogenic Neisseria have been infrequently characterized as they rarely cause systemic or life-threatening disease, except in immunocompromised individuals (18, 19).
Horizontal transfer and subsequent spread of commensal Neisseria resistance mechanisms has historically played a large role in rendering antibiotic therapies ineffective in Ngo. Reduced drug susceptibility in commensal Neisseria populations has been shown to be directly selected for after antibiotic usage (20), and thus these species will always be a persistent threat for resistance donation. In a U.S. gonococcal population dataset, over 11% of reduced susceptibility to azithromycin was acquired through inheritance of commensal transferable efflux pump (mtr) alleles (8-10). Additionally, the majority of resistance to third-generation cephalosporins in gonococci is derived through mosaic penicillin-binding protein 2 (penA) alleles gained from close commensal relatives (6, 10). Though studies that characterize the resistance genotypes and phenotypes in panels of commensal Neisseria (such as (17, 21, 22)) will aid in prospective surveillance for novel resistance that may be rapidly acquired by Ngo, the utility of these approaches is ultimately limited by extensive under sampling of natural commensal populations.
Experimental evolution can be leveraged as an alternative to phenotyping and genotyping large panels of bacteria, as a powerful screen for the genetic adaptations that underly antibiotic resistance in the commensal Neisseria, and thus may be at risk of HGT to Ngo. Laboratory evolution experiments reveal the spontaneous mutations caused by DNA replication and repair errors which increase mean fitness in new selective environments (23), and are ideal to illuminate the mechanisms of adaptation in bacteria due to their short generation times. In vitro selection has previously been used successfully to nominate mechanisms underlying ceftriaxone and azithromycin reduced susceptibility in Ngo (24, 25), and thus is a promising tool for illuminating new mechanisms of resistance in Neisseria commensals.
In this study, we use experimental evolution to investigate the potential for a Neisseria commensal (N. elongata, Nel) to evolve resistance to the macrolide antibiotic azithromycin, which until this year (26) was recommended as a first-line antibiotic, in combination with ceftriaxone, for the treatment of uncomplicated cases of gonorrhea in the United States. Here, we characterize the evolutionary response of Nel replicate lineages to azithromycin and consider if there is a single or multiple adaptive solutions to selection. Furthermore, we assess the fitness costs of derived mutations, and their potential for transfer into Ngo.
Results
Evolutionary trajectories to macrolide resistance in N. elongata
N. elongata AR Bank #0945 was selected to explore the evolutionary paths to azithromycin resistance in a Neisseria commensal. N. elongata AR Bank #0945 has been tested for its minimum inhibitory concentration (MIC) to azithromycin (0.38 μg/mL) and sequenced (SAMN15454046) by our group previously (21). In this study, a draft genome was assembled as an ancestral reference for all derived lineages. The assembly contained 2,572,594 bps, and 2,509 annotated genes (JAFEUH000000000).
Selective pressure was applied to sixteen replicates of AR Bank #0945 by creating a concentration gradient of azithromycin on standard growth media via application of Etest strips (Figure 1A). Cells were selected for passaging by sweeping the entire zone of inhibition (ZOI) and a 1 cm band in the bacterial lawn adjacent to the ZOI (Figure 1A). After 20 days, or approximately 480 generations, the average MIC value for all evolved cell populations increased to 7.6 μg/mL, which was significantly higher compared to day one values (W = 98.5, P = 0.0004), and ranged from 0.19 to 48 μg/mL (Figure 1B and C). Control populations with no drug selection (n=4), showed no increase in MIC compared to the ancestral stock. To mitigate the possibility of heterogenous cultures at the termination of the experiment, a single colony was selected from each evolved and control population for further MIC testing and genomic sequencing. MIC values for drug-selected single colony picks tended to be higher than those recorded for population values, and ranged from 0.5 to 64 μg/mL, with an average value of 14.4 μg/mL (Table 1).
Evolved cell lines were sequenced and aligned to the N. elongata AR Bank #0945 draft assembly to nominate derived polymorphisms. Mutations that were shared with control strains, or those shared with ancestral reads mapped back to the reference assembly, were not further considered. In total, 37 derived mutations were identified across all sequenced strains (Table 2). The most frequently observed mutations were found in the glucokinase encoded by glk (n=13 lineages), followed by those in rpmH encoding the 50S ribosomal protein L34 (n=4), and mutations in the intergenic region proximal to rpsC encoding the 30S ribosomal S3 protein (n=3). Unique mutations observed within annotated genes, were present in the coding domains for: the capsular polysaccharide phosphotransferase (encoded by cps12A), isocitrate lyase (aceA), RNA 2’-phosphotransferase (kptA), the di-/tripeptide transporter (dptT), and the bifunctional (p)ppGpp synthase/hydrolase (spoT). All strains with azithromycin MIC values ≥ 2 μg/mL (the Clinical & Laboratory Standards Institute (CLSI) reduced susceptibility breakpoint for N. gonorrhoeae (27)) were associated inheritance of mutations in either rpmH or the intergenic region proximal to rpsC.
Confirmation of the causality of high-level resistance encoding mutations
To assess the causality of the mutations encoding macrolide resistance in N. elongata AR Bank #0945, the ancestral stock was transformed with genomic DNA from evolved cell lines with MIC values ≥ 2 μg/mL (n=7). Genomic DNA from evolved lineages successfully transformed the ancestral stock in all cases. To identify causal loci, three colonies from each transformation were selected to characterize the polymorphisms which had been inherited from donor strains that were not present in the AR Bank #0945 ancestral recipient. The only region inherited across replicate transformant colony picks contained either mutations in rpmH (DNA donated from AM1, CBW4, JA1, or LT1), or the intergenic region near rpsC (DNA donated from CBW6, LJT1, or MRS1) (Table 2). Sanger sequencing confirmed the identity and presence of these mutations (Figure 2), and translation of rpmH duplications at the amino acid level indicated the in-frame insertions: 8SVTKRKRT15, 7LKRTYQ12, and 8HIMKRTYQ15 (Figure 3).
In most cases recovered transformants had azithromycin MICs that perfectly mirrored the donor strain phenotypes. However, all three transformants with CBW4 as a donor consistently had phenotypes of 48 μg/mL, one dilution below the donor strain phenotype of 64 μg/mL (Table 1). Additionally, LJT1 transformants had MIC values of 12 μg/mL, also one dilution below the donor strain phenotype (Table 1). Finally, one of the three AM1 transformants (T-AM1-3) had a lower MIC (12 μg/mL) than the other transformants and the AM1 donor strain (24 μg/mL).
Most novel ribosomal variants reduce in vitro fitness
In order to evaluate the fitness costs of azithromycin resistance-conferring mutations, the optical densities of transformant cells lines were compared to the ancestral N. elongata AR Bank #0945 strain over a 21-hour period (Figure 4A). At hour 21, OD600 values for AR-0945 replicates ranged from 0.68 to 0.81 (n=6); and six of the seven transformants had significantly lower optical densities (Figure 4B; Tukey’s HSD, p < 0.001). OD600 values for T-LJT-1 however ranged from 0.70-0.78 (n=6), which were not significantly different compared to the ancestral strain (Figure 4B; Tukey’s HSD, p < 0.99).
N. elongata ribosomal variants are inefficient or incapable of transfer to a pathogenic relative
Neisseria commensals are known sources of resistance for Ngo (6, 8-10), and therefore we test the ability of the evolved ribosomal duplication mutations uncovered in this study to be transferred into multiple Ngo strains. To establish a baseline rate of transformation for these alleles, we first quantify the number of transformants recovered for N. elongata AR Bank #0945 transformed with genomic DNA from evolved cell lines. In brief, we provided 100 ng of genomic DNA from evolved lineages in AR Bank #0945 cell suspensions, and after a 4-hour period allowing for the expression of any newly acquired alleles, selected on 4 μg/mL azithromycin. The percentage of colonies recovered on selective media as compared to colonies recovered on non-selective media was then calculated across three replicate trials, and averaged between 0.0003 to 0.0017 percent for all DNA (Figure 5).
Though in natural populations of Neisseria HGT from commensals to Ngo has been repeatedly demonstrated, recent work has shown that commensal DNA is toxic to Ngo (28, 29). Ngo uptakes DNA through a Type IV pilus (Tfp)-based system, which binds preferentially to Neisseria-specific DNA (1-3). This DNA is then transported across the cell wall, and becomes integrated via homologous recombination into the genome by RecA. However, since Nel and Ngo have different intrinsic methylases, Ngo restriction enzyme(s) cleave incorporated DNA at heteroduplexes with Nel methylation signatures, resulting in the loss of chromosome integrity and cell death (28, 29). Nel DNA has been shown to be rendered less toxic through in vitro methylation using M.CviPI and M.SssI methyltransferases to modify cytosines in CpG and GpC motifs (28, 29).
Thus, transformation of Nel DNA was attempted with methylation modification (via M.CviPI and M.SssI methyltransferases) into naturally competent pilated Ngo 28Bl and FA1090 stocks. In all cases we recovered no colonies on selective media (Figure 5), following the same transformation protocol as used for Nel within this study, and which we have used previously for transformation of Ngo with Ngo DNA (8).
Discussion
Multiple studies have demonstrated that commensal Neisseria serve as reservoirs of resistance for Ngo (6, 8-10), however a comprehensive evaluation of the resistance alleles commensals can harbor, though underway (17, 21, 22), and their likelihood of transfer to pathogenic relatives is still in its infancy. Here, we use experimental evolution to screen for the mutations that impart azithromycin resistance in Nel and assess their potential for transfer to Ngo.
Overall, our results support constrained evolutionary trajectories to high-level macrolide resistance in Nel. A diversity of azithromycin resistance mutations have been reported in Ngo, including: alterations in the 23S rRNA azithromycin binding sites (C2611T and A2059G) (10, 30, 31), a G70D mutation in the RplD 50S ribosomal protein L4 (32), rplV tandem duplications (10), variants of the rRNA methylase genes ermC and ermB (33), and mutations that enhance the expression of Mtr or increase the binding efficiency of MtrD with its substrates (8, 9, 34-36); however, high-level resistance is most frequently imparted by the aforementioned ribosomal mutations (10, 32). Similarly, for all cases of high-level resistance emergence in Nel drug-selected lineages within this study, causal mutations converged on either tandem duplications in the gene encoding the 50S ribosomal L34 protein (rpmH) or the intergenic region proximal to the 30S ribosomal S3 protein (rpsC).
Fascinatingly, the evolved L34 mutations in Nel are identical or nearly identical to those recently reported in Ngo. High-level resistance in Ngo was found to be conferred by the L34 duplications 7PSVTKRKR14, 7PSVTNTYQP14, and 7LKRTYQ12 (25); and occurred in the same location as the Nel duplications 8SVTKRKRT15, 8HIMKRTYQ15, and 7LKRTYQ12 uncovered in this study (Figure 3). Preserved identity and location of rpmH mutations across Neisseria species suggests that this may be a conserved mechanism of rapid macrolide resistance acquisition across the genus. Though we do not find any of the other Ngo mutations in ribosomal components (C2611T, A2059G, rplD G70D, or rplV tandem duplications) in Nel, we describe novel azithromycin resistance conferring mutations in the intergenic region upstream of rpsC, similarly, produced through tandem duplication events (Figure 2). To our knowledge these have not yet been reported in Neisseria, and likely impact the expression of rpsC due to their location in or proximal to the promoter.
Almost all of the tandem duplications reported here imparted some fitness cost, as measured by in vitro growth assays of isogenic cell lines (Figure 4). Ngo duplication mutations in L34 were reported to be transitory and repeatedly lost in culture (25), further suggesting a fitness cost in alternate genetic backgrounds. However, the rpsC duplication in T-LJT-1 appeared to have no impact on fitness (Figure 4), suggesting that it may be sustained in natural populations. Further work will be needed to elucidate the long-term stability of all uncovered mutations, and to assess if they are either transitory stepping-stones or persistent, when coupled with compensatory mutations (e.g., as is the case for a variant in acnB mitigating growth defects in ceftriaxone resistant penA mutants (37)), mechanisms of macrolide resistance in commensal Neisseria species.
Assessing the likelihood of commensal resistance alleles to be transferred to Ngo will aid in determining those resistance mechanisms most at risk of rapid dissemination into pathogen populations; and may guide future prospective genotyping practices during routine public health surveillance efforts. Here, we attempted to transform novel Nel alleles into Ngo genetic backgrounds, however we were unable to recover transformants in all cases (Figure 5). One explanation for failed transformations could be insufficient sequence homology for RecA-mediated homologous recombination. Though, this is unlikely as rpmH shares 87% nucleotide and 100% amino acid identity between N. elongata AR Bank #0945 and to both Ngo FA1090 and 28Bl; similar to the percent identity of mosaic mtr sequences (∼90%) which we were previously able to transform into the 28Bl background (8). The sequence region including rpsC and 200 bps upstream of the start site is more divergent however, and only shares 78% identity between AR-0945 and the Ngo strains used in this study, which is perhaps sufficiently divergent to preclude HGT. Alternative explanations for failed HGT could include: an insufficient amount of donor DNA was provided, DNA methylation using M.CviPI and M.SssI methyltransferases was unsuccessful (28, 29), incompatible DNA uptake sequences (DUSs) (3), or incompatible genomic backgrounds for the expression of these resistance mechanisms; however, follow up studies will be needed to discriminate the underlying cause(s).
Overall, our results expand on prior studies that provide initial insights into illuminating the commensal resistome (17, 21, 22), which is a known source of antibiotic resistance for Neisseria pathogens (6, 8-10). We find evidence of constraint on high level macrolide resistance genotypic evolution in AR-0945, with convergence on only two sites in the genome, however different genetic starting places will likely impact evolutionary outcome due to epistatic and additive effects between loci. Thus, future work will not only focus on expanding this approach to other commensal species and therapeutics, but will incorporate intraspecific variation as an additional consideration. Ultimately, this work emphasizes the power of experimental evolution in characterizing the genetic pathways to resistance in commensals species, which will be key to illuminating mutations at risk of transfer across species boundaries and their effects.
Methods
Bacterial strains and growth conditions
The bacterial strain N. elongata AR Bank #0945 used for this study was obtained from the Centers for Disease Control and Prevention (CDC) and Food and Drug Association’s (FDA) Antibiotic Resistance (AR) Isolate Bank “Neisseria species MALDI-TOF Verification panel”. For all experiments, N. elongata AR Bank #0945 and its evolved derivatives were revived from trypticase soy broth (TSB) stocks containing 50% glycerol stored at -80°C. Stocks were streaked onto GC agar base (Becton Dickinson Co., Franklin Lakes, NJ, USA) media plates containing 1% Kelloggs solution (38) (GCP-K plates), and were grown for 18-24 hours at 37°C in a 5% CO2 atmosphere.
Experimental evolution was conducted by passaging 16 replicate stocks of N. elongata AR Bank #0945 in the presence of azithromycin for 20 days or ∼480 generations. A selective gradient of azithromycin was applied to GCB-K plates using Etest strips (bioMérieux, Durham, NC), and each day overnight growth was collected from the entire zone of inhibition (ZOI) and a 1 cm region in the bacterial lawn surrounding the ZOI (Figure 1). Collected cells were suspended in TSB, and plated onto a fresh GCB-K plate with a new Etest strip. MICs each day were determined by reading the lowest concentration that inhibited growth, and reduced susceptibility was determined using the CLSI guidelines for N. gonorrhoeae (breakpoint AZI ≥ 2 μg/mL). MICs were read by at least two independent researchers. Five strains were passaged using the same protocol on media containing no selective agent. Final cell populations were streaked onto GCB-K plates and individual colonies were picked and stocked for further analysis.
Whole genome sequencing and comparative genomics
Cells from evolved cell lines were lysed by suspending growth from overnight plates in TE buffer (10 mM Tris [pH 8.0], 10 mM EDTA) with 0.5 mg/mL lysozyme and 3 mg/mL proteinase K (Sigma-Aldrich Corp., St. Louis, MO). DNA was purified using the PureLink Genomic DNA Mini kit (Thermo Fisher Corp., Waltham, MA) and treated with RNase A. Sequencing libraries were prepared using the Nextera XT kit (Illumina Corp., San Diego, CA), and uniquely dual-indexed and pooled. Each pool was subsequently sequenced the Illumina MiSeq platform at the Rochester Institute of Technology Genomics Core using V3 600 cycle cartridges (2×300bp). Sequencing quality of each paired-end read library was assessed using FastQC v0.11.9 (39). Trimmomatic v0.39 (40) was used to trim adapter sequences, and remove bases with phred quality score < 15 over a 4 bp sliding window. Reads < 36 bp long, or those missing a mate, were also removed from subsequent analysis. The AR-0945 draft assembly was constructed using SPAdes v.3.14.1 (41) and annotated with prokka v.1.14.5 (42). Trimmed reads were mapped back to the AR-0945 draft assembly using Bowtie2 v.2.2.4 (43) using the “end-to-end” and “very-sensitive” options and Pilon v.1.16 (44) was used to call variant sites. Only single nucleotide polymorphisms and short indels were retained, and any variants called within 100 bp of the end of contigs were removed.
Transformations
Transformations were conducted by inoculating GCP broth (7.5 g protease peptone 3, 0.5 g soluble starch, 2 g dibasic K2HPO4, 0.5 g monobasic KH2PO4, 2.5 g NaCl, and double-distilled water [ddH2O] to 500 ml; Becton Dickinson Co., Franklin Lakes, NJ) supplemented with 1% IsoVitaleX and 10 mM MgCl (Sigma-Aldrich Corp., St. Louis, MO) with cells to an optical density (OD) of ∼0.5. Cell suspensions were subsequently incubated with 100 ng of gDNA for 4 hours to allow DNA uptake, homologous recombination, and expressions of new alleles. Cell suspensions were then serially diluted to allow for quantification of transformation efficiency and spotted onto GCB-K plates containing 4 μg/mL of azithromycin. Cultures then were incubated overnight and after 18 hours colonies were counted for each reaction and azithromycin resistant transformants were selected by picking single colonies. Transformants were subsequently MIC tested and whole-genome sequenced to nominate inherited derived mutations.
Polymerase chain reaction (PCR) and Sanger sequencing were used to confirm the location and identity of all derived polymorphisms nominated in transformants from genomic sequencing screens. In brief, PCR was conducted in 50-μl volumes using Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA). Primers for rpmH (F: 5’-CGAAGCTTTCCAAAACGGCT-3’; R: 5’-AAGGTTCGGCCAAAGATTGC-3’) and the rpsC/rplB intergenic region (F: 5’-ATCGCTACTTTTAGCAAACCACT-3’; R: 5’-TGCAGAGCATAATGAAGGTGCT-3’) were annealed at 60°C. Reactions were conducted for 35 cycles, with 30 second extensions. Resultant products were cleaned using ExoSAP-IT (Applied Biosystems, Foster City, CA), and sequenced via the Sanger method.
For Ngo transformation experiments, recipient 28Bl and FA1090 cell stocks were obtained from the CDC and Yonatan Grad at the Harvard T.H. Chan School of Public Health respectively. These recipients were provided with genomic DNA from the evolved Nel strains generated within this study both with and without methylation modification. In brief, for modified DNA 500 ng of Nel DNA was incubated with M.CviPI and M.SssI methyltransferases (New England Biolabs, Ipswich, MA) as per the manufacturer’s instructions, and as specified in (29). 100 ng of modified Nel DNA was then provided to 28Bl and FA1090 as transformation substrate, following the aforementioned protocol.
Growth Curves
Cells were inoculated into GCP broth supplemented 1% Kelloggs solution to an OD600 of ∼0.1, using a Genesys 150 spectrophotometer (Thermo Scientific, Waltham, MA). Cell suspensions were subsequently distributed into 96-well plates and incubated at 37°C in a BioTek Synergy H1 microplate reader (BioTek, Winooski, VT). Subsequent OD600 measurements were taken every hour for 21 hours, with a 1-minute shake at 180cpm prior to reading. The BioTek Gen5 v.3.05 software was used to interpret OD values. Replicates were completed for each cell line (n=6), and all downstream analyses were performed in R (45).
Data Availability
All scripts and datasets are available on: https://github.com/wadsworthlab. The draft assembly for N. elongata AR Bank #0945 is deposited to GenBank (accession: JAFEUH000000000). Read libraries for the genomics datasets generated in this study can be accessed on the Sequence Read Archive for evolved strains (SAMN17958618-SAMN17958637) and transformants (SAMN18355358-SAMN18355369).
Acknowledgements
This work was produced by the members of the Fall 2020 Genomics course (BIOL340) at the Rochester Institute of Technology (RIT) – a big thank you to each and every student for their dedication and hard work during an especially trying semester. The authors would like to acknowledge the generous support provided by the RIT College of Science for this study. The authors would also like to thank Narayan H. Wong and the RIT Genomics Core for providing sequencing support.