Rolling the evolutionary dice: Neisseria commensals as proxies for elucidating the underpinnings of antibiotic resistance mechanisms and evolution in human pathogens

ABSTRACT Species within the genus Neisseria are adept at sharing adaptive allelic variation, with commensal species repeatedly transferring resistance to their pathogenic relative Neisseria gonorrhoeae. However, resistance in commensals is infrequently characterized, limiting our ability to predict novel and potentially transferable resistance mechanisms that ultimately may become important clinically. Unique evolutionary starting places of each Neisseria species will have distinct genomic backgrounds, which may ultimately control the fate of evolving populations in response to selection as epistatic and additive interactions coerce lineages along divergent evolutionary trajectories. Alternatively, similar genetic content present across species due to shared ancestry may constrain existing adaptive solutions. Thus, identifying the paths to resistance across commensals may aid in characterizing the Neisseria resistome—or the reservoir of alleles within the genus as well as its depth. Here, we use in vitro evolution of four commensal species to investigate the potential and repeatability of resistance evolution to two antimicrobials, the macrolide azithromycin and the β-lactam penicillin. After 20 days of selection, commensals evolved resistance to penicillin and azithromycin in 11/16 and 12/16 cases, respectively. Almost all cases of resistance emergence converged on mutations within ribosomal components or the mtrRCDE efflux pump for azithromycin-based selection and mtrRCDE, penA, and rpoB for penicillin selection, thus supporting constrained adaptive solutions despite divergent evolutionary starting points across the genus for these particular drugs. Though drug-selected loci were limited, we do identify novel resistance-imparting mutations. Continuing to explore paths to resistance across different experimental conditions and genomic backgrounds, which could shunt evolution down alternative evolutionary trajectories, will ultimately flesh out the full Neisseria resistome. IMPORTANCE Neisseria gonorrhoeae is a global threat to public health due to its rapid acquisition of antibiotic resistance to all first-line treatments. Recent work has documented that alleles acquired from close commensal relatives have played a large role in the emergence of resistance to macrolides and beta-lactams within gonococcal populations. However, commensals have been relatively underexplored for the resistance genotypes they may harbor. This leaves a gap in our understanding of resistance that could be rapidly acquired by the gonococcus through a known highway of horizontal gene exchange. Here, we characterize resistance mechanisms that can emerge in commensal Neisseria populations via in vitro selection to multiple antimicrobials and begin to define the number of paths to resistance. This study, and other similar works, may ultimately aid both surveillance efforts and clinical diagnostic development by nominating novel and conserved resistance mechanisms that may be at risk of rapid dissemination to pathogen populations.

T he emergence of antibiotic resistance within bacterial populations is mediated by natural selection, whereby mutations encoding drug-protective mechanisms are produced stochastically and subsequently increase in frequency as a result of only the cells harboring these mutations surviving exposure events.However, a key ques tion for both understanding the evolutionary process and also the enhancement of surveillance efforts is: how repeatable and predictable is resistance evolution at the genotypic level?Two alternate hypotheses can be advanced: (i) adaptive landscapes are constrained to one or few solutions (i.e., genotypic constraint) or (ii) a multitude of fitness peaks exist created by many mutations imparting similar phenotypic outcomes.Many prior studies support some level of genotypic constraint on resistance evolution at the strain or species level (1-5); however, less frequently has the repeatability of resistance evolution been interrogated across species' boundaries.Applying selection across different genomic backgrounds at the species level may lead us to predict a higher likelihood of divergent evolutionary outcomes, with different mutations giving rise to similar phenotypic resistance in different species.We may predict this given that the pre-existing suite of potentially additive and/or epistatically interacting mutations already present in each species' genomes will likely be unique as a result of both genetic drift since the time of lineage divergence and also niche-specific adaptation.However, if genotypic convergence is observed across species, this suggests constrained ranges of adaptive solutions between high-level taxonomic groupings (e.g., genera, families, etc.) due to their shared ancestral history and conserved genetic makeup.Here, we begin to interrogate this question: does genotypic constraint or divergence govern the emergence of resistance evolution within the genus Neisseria?
The genus Neisseria is composed of several Gram-negative, typically diplococcoid, oxidase-positive, and often catalase-positive species, which most frequently colonize the nasopharyngeal or oral niche in humans or animals (6).There are, at a minimum, 10 human-associated species that likely evolved from a common ancestor that colonized an early humanoid (7).Interestingly, contemporary Neisseria has distinct colonization sites within the oro-and nasopharynx suggesting ecological and physiological divergence between species since that initial colonization event (8).Some species are carried harmlessly as commensals in 100% of healthy human adults and children (Neisseria cinerea, Neisseria polysaccharea, Neisseria lactamica, Neisseria mucosa, Neisseria oralis, Neisseria subflava, Neisseria elongata [atypical rod], and Neisseria bacilliformis [atypical rod]); however, two species have significant pathogenic potential (Neisseria gonorrhoeae and Neisseria meningitidis) and are carried in a smaller percentage of the population (between 0.01% and 10%) (9)(10)(11)(12)(13).N. gonorrhoeae is unique within the genus in that in addition to colonizing the nasopharyngeal mucosa, it also routinely colonizes the urogenital tract and rectum and causes the sexually transmitted infection gonorrhea (14).
Though the genetic basis of some resistance phenotypes appears to be exclusively encoded by recurrently acquired mutations {i.e., ciprofloxacin resistance is almost always caused by amino acid substitutions in the DNA gyrase subunit A (GyrA S91F and D95G/D95A [24,25])}, the complete genetic bases of other resistance phenotypes are currently not fully described and/or imparted by additive or epistatically interacting loci for which the combined effects must be quantified (e.g., penicillin [26][27][28][29][30] and azithromycin [25,31] resistance).For example, known mutations decreasing susceptibil ity to penicillin have been described in: penA (encoding penicillin-binding protein 2 [PBP2]) (32), ponA (penicillin-binding protein 1 [PBP1] (28), Mtr efflux pump components (29), the major outer membrane porin protein P1B allele (26), and β-lactamase harboring plasmids (33); however, at least one unknown untransformable resistance determinant exists ("factor X") (34).In addition, most of the described azithromycin resistance can be gained through mutations in Mtr efflux pump components (35), mutations in the 23S rRNA azithromycin-binding sites (C2611T and A2059G) (36,37), and ribosomal protein mutations (38).However, a large proportion of lower-level resistance remains unexplained (25,38).Thus, experimentally interrogating the paths to resistance and their repeatability in Neisseria will become an important step for identifying novel contributing mutations, identifying the combinations of loci that contribute to polygenic resistance, and understanding their potential prevalence and evolution within popula tions.
Studies on the paths to resistance within gonococci have previously been explored in vitro (39)(40)(41)(42)(43)(44).However, gonococci, in addition to gaining resistance through de novo mutations, are also superbly adept at acquiring resistance from their close commensal relatives (5,31,(45)(46)(47).This allelic exchange across Neisseria species likely occurs in their shared colonization sites of the naso-and oropharyngeal niches (8), with the whole genus often being referred to as a consortium with "fuzzy" borders due to the high frequency of DNA donation through horizontal gene transfer (48)(49)(50).Commensal species thus serve as a bubbling cauldron of new adaptive solutions and reservoirs of resistance for gonococci, with each species containing a unique genomic background in which novel resistance genotypes may emerge.Therefore, expanding the investiga tion on the repeatability of evolution to the entire genus may serve two important goals in the fight against the spread of resistance in gonococci: (i) identifying resist ance phenotypes for which a multitude of genotypic paths exist, either within distinct genomic contexts or across several and (ii) determining which drugs and/or drug classes have limited adaptive solutions within the genus.Both of these findings may guide the development of nucleic acid-based resistance tests [i.e., nucleic acid-based resistance tests (NAAT) or whole genome sequencing (WGS)] for surveillance programs by defining the scope of mutations that must be surveyed.
Here, we begin to interrogate the paths to resistance to two drugs with as-of-yet not fully identified genotypic bases within the pathogenic Neisseria.We use four different genomic contexts across the Neisseria genus (N.cinerea, N. subflava, N. elongata, and Neisseria canis) and select for increasing minimum inhibitory concentrations (MICs) by passing each species across selective gradients as previously described (5).Though the scope of this initial and a prior study (5) has been limited (i.e., limited species and experimental replicates), we imagine that by continuing to "roll the evolutionary dice" we will ultimately coalesce on the possible paths to resistance and their quantity, addressing the repeatability of evolution to different drug classes across the genus.Finally, both this and our previous study (5) were conducted as part of exercises within undergrad uate classrooms at the Rochester Institute of Technology, highlighting the power of experimental evolution in addressing fundamental questions impacting global public health, while also providing important experiential learning opportunities for entry-level students.

The frequency and identity of derived mutations
For each evolved lineage, a single colony was picked for further characterization and whole-genome sequencing (Table S1).There were no significant differences between the number of derived mutations after the 20-day long experiment between drugs across all species; however, each species and interaction between drugs and species (two-way ANOVA: P = 0.0008) had a significant and nearly significant (two-way ANOVA: P = 0.055) impact on the number of derived mutations, respectively.N. elongata had significantly fewer derived mutations compared to N. canis (Tukey's HSD: P = 0.02), N. cinerea (Tukey's HSD: P = 0.0007), and N. subflava (Tukey's HSD: P = 0.004).Differences in the number of derived mutations did not appear to be correlated to growth rate as doubling times were calculated at 224 minutes for N. cinerea, 258 minutes for N. elongata, 299 minutes for N. canis, and 715 minutes for N. subflava (Fig. S1), which may support different baseline mutation rates between species.While Neisseria pathogens are reported to divide more rapidly (40-60 minutes), longer division times have been reported for commensals previously (56).When separated by drug class, for penicillin, both N. canis and N. cinerea had significantly more derived mutations compared to N. elongata (Tukey's HSD: P = 0.02 and P = 0.059 respectively; Fig. 4), and for azithromycin, N. subflava had significantly more novel mutations compared to N. elongata (Tukey's HSD: P = 0.045; Fig. 4).Mutations within coding domain sequences (CDSs) and rRNA sequences were identified for all evolved lineages and, after correcting for mutations also present in control lineages with no drug exposure, were considered candidates for imparting resistance (Fig. 5; Table 2).For azithromycin, all replicate lineages of N. subflava, N. canis, and N. cinerea evolved resistance; however, none of the N. elongata strains did (Fig. 1).The most frequent mutation occurring in N. subflava lineages was located within pilM, a component of type IV pili (Tfp).Additional mutations that emerged included those in beta-ketoacyl-ACP synthase III (encoded by fabH), a Maf-family transcription factor (mafB5), NADH-quinone-oxidoreductase (nqr), the 50S ribosomal protein L16 (rplP), the 50S ribosomal protein L22 (rplV), and the 50S ribosomal L34 protein (rpmH).For N.canis, the most frequent mutations occurred in the repressor of the Mtr efflux pump (mtrR), followed by rplV, duf2169, the inner membrane component of the Mtr efflux pump (mtrD), and a component of the glycan biosynthesis pathway (pglB2).Finally, for N. cinerea, mutations emerged in glucokinase (glk), ribosomal protein L11 methyltransfer ase (prmA), and rpmH.For penicillin, all replicate evolved lineages gained resistance except for one N. elongata strain and all N.subflava strains; however, each of these lineages developed increased MICs compared to the ancestral strains and had MICs ≥ 1 µg/mL.Mutations in N. subflava lineages that emerged include those in mtrD and mtrR, elongation factor Tu (tufA), and the endolytic murein transglycosylase (mltG).The most frequent mutations in N. canis include those in the 16S and 23S rRNAs, followed by those in PNL71104_P2, phosphoheptose isomerase (gmhA), an HTH11-domain coding protein, a phage-associated protein, peptide chain release factor 2 (prfB), DNA-direc ted RNA polymerase subunit alpha (rpoA), and tRNA-fMet(cat).In N. elongata, derived mutations include those in mtrD, PBP2 (penA), 4-diphosphocytidyl-2-C-methyl-D-erythri tol kinase (ispE), and queuine tRNA-ribosyltransferase (tgt).Finally in N. cinerea, mutations included those in penA, pilM, glk, phosphate transporter (pitA), exopolyphosphatase (ppx), DNA-directed RNA polymerase subunit beta (rpoB), and HTH-type protein slmA (slmA), along with some additional singleton mutations (Fig. 5).

DISCUSSION
Commensal Neisseria has repeatedly donated resistance alleles to their pathogenic relative N. gonorrhoeae (31,(45)(46)(47) and beyond doubt serve as a bubbling cauldron of new adaptive solutions to address "the antibiotic crisis" that N. gonorrhoeae faces.However, we do not yet understand the full suite of resistance alleles that commensal Neisseria can carry, if the pool of mechanisms is large or small and if the pool size varies by antibiotic.
Here, we roll the evolutionary dice using antibiotic selection across divergent commensal Neisseria genomic contexts to begin to answer three important questions: (i) what are the identities of resistance mutations that emerge in commensals in response to selection, (ii) are the paths to resistance constrained or diverse, and (iii) do resistance mutation identities and paths vary by drug class?Azithromycin is a macrolide antibiotic that inhibits protein synthesis by binding to the 23S rRNA component of the 50S ribosome.Mutations that impact the conformation or block the binding site of the drug have previously been described in N. gonorrhoeae to impart resistance and include mutations in the 23S rRNA azithromycin-binding sites (C2611T and A2059G) (36,57), a G70D mutation in the RplD 50S ribosomal protein L4 38 , and rplV tandem duplications (25).Here, we find a suite of variants that emerged post-selection within the CDSs encoding ribosomal proteins.For example, in both N. subflava and N. canis, we uncovered mutations emerging in rplV encoding the 50S ribosomal protein L22, with 2/4 N. subflava lineages (78-KGPSLKRFQARA) and 2/4 N. canis (91-KRFQARAKG and 91-GRGNRIQARAKG) lineages evolving tandem duplications or insertions within this gene.Similar duplications in this region have previously been predicted to block the azithromycin-binding site in N. gonorrhoeae (25).In-frame insertions in rpmH, which encodes the 50S ribosomal L34 protein, were also frequent and found within 2/2 surviving N. cinerea and 2/4 N. subflava strains.N. cinerea strains both evolved distinct rpmH variants (6-HHETHLS and 6-QLMKRTYQ), while N. subflava strains evolved the same variant (8-SVKRTYQP).Similar duplications in rpmH have been previously described in N. elongata (5) and N. gonorrhoeae (40) and have been found to be casual to high-level azithromycin resistance through a transformation in N. elongata (5).Thus, these are the likely mutations imparting high-level resistance in N. cinerea strains within this study.Interestingly, the N. elongata strains evolved in this study did not evolve reduced azithromycin susceptibility (Fig. 1; Table 1); however, in our prior work (5), only 44% of replicate N. elongata lineages evolved resistance, and only 43% of these resistant isolates gained resistance through mutations in rpmH.With only four replicate N. elongata strains selected in this study, we speculate that we did not have sufficient power to uncover rpmH variants in N. elongata, as we have previously found they have a slower growth rates (5) and, therefore, may have been outcompeted.Finally, we find evidence for a duplication within the rplP gene encoding the 50S ribosomal protein L16 within a single N. subflava strain; however, we find no difference in MICs between this strain which also harbors a rplV duplication and a second strain with just a rplV duplication, suggesting that the variant uncovered in rplP may not contribute to the elevated MICs observed.Manoharan-Basil et al. (2021) (69) describe multiple recombina tion events in genes encoding ribosomal proteins across pathogenic and commensal Neisseria, supporting the possibility of transfer of these types of resistance mutations in natural Neisseria populations.
The multiple transferable resistance efflux pump (Mtr) is a primary mechanism by which N. gonorrhoeae gains resistance to both azithromycin and penicillin.The Mtr efflux pump is composed of the MtrC-MtrD-MtrE cell envelope proteins, which together export diverse hydrophobic antimicrobial agents, such as antibiotics, nonionic detergents, antibacterial peptides, bile salts, and gonadal steroidal hormones from the cell (70)(71)(72)(73).Overexpression of the pump, through mutations that ablate or decrease the expression of the repressor of the pump (MtrR), has been demonstrated to increase resistance to both azithromycin and penicillin (25,29,74,75), and substitutions within the inner membrane component MtrD have been shown to decrease susceptibility to azithromy cin (31,46).Here, in response to azithromycin-based selection, all four experimental replicates of N. canis evolved mutations in MtrR: two with a G172D substitution, one A37V, and one insertion impacting the reading frame and resulting in a premature stop codon.Then, 3/4 replicates of N. subflava evolved mtrR mutations in response to penicillin exposure which resulted in a T11I substitution in MtrR.MtrR mutations uncovered in commensals within the study have not been described in N. gonorrhoeae to impart azithromycin-reduced susceptibility previously; however, the A37V mutation in N. canis is proximal to the predicted helix-turn-helix domain of the MtrR protein known to be important in DNA-binding activity in N. gonorrhoeae (59).MtrD mutations also emerged in response to penicillin-selection in N. subflava (L996I) and N. elongata (with all three strains carrying different mutations: V139G, F604I, or A1009T).Finally, a mutation encoding MtrD E823K also emerged in 1/4 N. canis strains after azithromycin selection.Interestingly, this last E823K MtrD substitution was predicted to be the causal mutation in mosaic commensal Neisseria alleles imparting azithromycin resistance and transferred to N. gonorrhoeae (31,46).β-lactams, such as penicillin, target the penicillin-binding proteins and inhibit cell wall biosynthesis.Mutations in PBP2 (encoded by penA) in particular have been well documented to impart elevated penicillin MICs in N. gonorrhoeae (28,76) and also other β-lactams including the extended spectrum cephalosporin ceftriaxone, through both native gonococcal alleles (77) and non-native alleles acquired from commensal Neisseria (25,47,76,78).These mutations act by lowering the affinity of the beta-lactam antibiot ics for PBP2 and also by restricting the motions of PBP2 which are important for acylation by beta-lactams (79).Therefore, unsurprisingly, we observed multiple mutations emerge in penA, though only in two species: N. elongata and N. cinerea.Then, 3/3 surviving N. elongata evolved lines had penA mutations emerge: P399S, V574E, and A581S; all four experimental N. cinerea replicates evolved penA mutations encoding the amino acid substitutions: F518S, V548E, and A549E.All uncovered PenA mutations emerged in the transpeptidase domain of the protein (80).Mutations in another penicillin-binding protein, PBP1 encoded by ponA, also contribute to reduced penicillin susceptibility in gonococci (28); however, we did not observe the emergence of any ponA mutations within the commensal Neisseria evolved within this study.
Additional derived mutations of note that emerged after selection include those in the RNA polymerase and components of the pilus.Here, after penicillin selection, a rpoA mutation emerged in N. canis, and rpoB mutations emerged in N. cinerea.In N. gonorrhoeae, both RpoD (E98K and Δ92) and RpoB (R201H) mutations impact ceftriax one susceptibility, another β-lactam antibiotic, likely through increased expression of PBP1 and reduced expression of D,D-carboxypeptidase (68).Here, the rpoA G147A nucleotide substitution in N. canis resulted in a silent change, so it does not likely contribute to elevated penicillin MICs; however, evolved rpoB mutations encoded amino acid substitutions (E345A and P591S) in 2/4 N. cinerea replicate lineages.Though these particular rpoB mutations have not been demonstrated to impart resistance in Neisseria previously, associations between mutations within this locus and ceftriaxone-reduced susceptibility in N. gonorrhoeae (68) suggest that it may be important to further explore the relationship between new rpoB variants and resistance to β-lactam antibiotics.Another locus with a derived mutation that may reduce susceptibility to β-lactams is the lytic transglycosylase mltG.MltG acts as a terminase, ceasing peptidoglycan strand growth through cleavage of the growing glycan, and its deletion has been shown to increase susceptibility to penicillin in N. gonorrhoeae (81).In this study, a mutation emerged in a single N. subflava lineage in response to penicillin selection which resulted in a P293L substitution.Though rare, it may be an interesting candidate to further investigate with its known involvement in cell wall biosynthesis processes; however, its presence did not raise base MIC values from the ancestral strain, suggesting it has a marginal impact on resistance (if any).Finally, the pilus-associated mutations in PilM in N. cinerea in response to penicillin selection (4/4 isolates; 2 with a 1-bp deletion at position 404, and 2 with a 1-bp deletion at position 162) and azithromycin selection (1/4 isolates; 1 bp deletion at position 404), and PilQ in N. subflava (a 1 bp deletion at position 593 in 4/4 strains) in response to azithromycin all shift the reading frames resulting in non-function proteins.These mutations likely impact drug diffusion across the outer membrane and into the periplasm in some way similar to gonococci (82) (i.e., modification of the structure or assembly of the multimeric PilQ pore complex).Pilus-associated mutations may be important mechanisms of antibiotic escape for commensal species as there is some evidence that they do not require type IV pili for host-cell attachment (83); however, they may have a high fitness cost in Neisseria pathogens which do require functional pili (84) and thus are not likely candidates for long-term persistence in N. gonorrhoeae or N. meningitidis populations.
When investigating the haplotypes of each evolved strain, variants contributing to elevated MICs became more apparent in some cases (Table S4).For azithromycin selection, N. canis evolved MtrR mutations in all evolved replicates; however, only the addition of MtrD E823K or RplV tandem duplications was sufficient to elevate MICs > 16 µg/mL (the base MIC after one MtrR mutation was acquired).For N. subflava, duplications in RplV increased MICs more than those in RpmH (256 µg/mL vs 96 µg/mL, respectively).For penicillin-based selection in N. cinerea, the PenA A549E mutation had the largest impact on elevated MICs compared to the other PenA mutations that emerged (V548E and F518S).For N. cinerea as well, the RpoB P591S alone raised MICs from 2 µg/mL to 6 µg/mL.For N. canis, though all strains evolved resistance through acquisition of PenA and MtrD mutations, the haplotype with the highest MIC was PenA V574E and MtrD V139G (12 µg/mL compared to ≤2 µg/mL).Finally, in reviewing ancestral strains with above breakpoint MICs (Table 1), some mutations that were evolved in this study or identified previously were present that may further support their involvement in reduced susceptibility.For example, in the ancestral N. cinerea, we find MtrR A39T and G172D.However, in the ancestral N. subflava strains, we do not find described resistance mutations, suggesting there are more contributors to be identified.
The aforementioned ribosomal, MtrRCDE, PenA, RpoB, and pilus-associated mechanisms seem to be the likely contributors to the emergence of reduced suscept ibly in all of the Neisseria commensals investigated in this study for both penicillin and azithromycin-based selection (Fig. 6).Therefore, despite 2/2 N. canis replicates evolving low-level penicillin resistance with as-of-yet unexplained genetic bases, with 19/21 cases of Neisseria evolution converging on known resistance modalities descri bed in other Neisseria species (25,68,82), we must accept a constrained range of adaptive solutions to antibiotic selection within the genus at this point.The remaining questions do exist, however.For example, here, we primarily investigate coding-domain regions; thus, important mutations in intergenic regions such as promoters could have been missed.However, we specifically searched for mutations within the mtrR-CDE and macAB promoter regions because known resistance-conferring mutations within the regions have been identified previously (29, 60, 85) yet we found none within this study.We also acknowledge that our small sample of strains and experimental replicates, and subsampling of each evolved lineage by selecting a single colony, may have limited the pool of potential resistance mechanisms uncovered.For example, some mechanisms may be less frequently observed due to high fitness costs, necessitating the evolution of compensatory mutations.These types of mutations may, therefore, be missed in small-scale experimental studies.Finally, evolution does not occur in controlled laboratory environments, so what is the role of intergenus gene exchange in Neisseria resistance emergence?For example, the β-lactamase-containing plasmid pbla present within the gonococcal and meningococcal populations was acquired from the patho genic Haemophilus ducreyi (86,87).Therefore, are there other clinically relevant resistance mechanisms available to the Neisseria that could be acquired from other inhabitants of the nasopharyngeal mucosa, urogenital tract, or rectum [see references (45) for additional discussion]?In summary, our current results highlight conserved paths to resistance within the Neisseria genus, though continued tosses of the evolutionary dice across different drug classes (e.g., tetracyclines, quinolones, etc.) and strain combinations may ultimately paint a different picture which will be the focus of future experiential learning opportunities within undergraduate classrooms at RIT.

Experimental evolution, MIC testing, and growth curves
MICs were measured by Etest strips (bioMérieux, Durham, NC) on GCB-K plates according to the manufacturer's specifications.In brief, cells from overnight plates were suspen ded in TSB to a 0.5 McFarland standard, and 200 µL of the suspension subsequently inoculated onto GCB-K plates.Etest strips were incubated on these plates for 18-24 hours at 37°C in a 5% CO 2 incubator.MICs were subsequently determined by reading the lowest concentration that inhibited the growth of bacterial lawns.Etests have been demonstrated to report equivalent MICs to the agar-dilution method for Neisseria (88).
For each of the four Neisseria sp.used in the study, four replicates were passaged on GCB-K plates containing a selective gradient of either penicillin or azithromycin.Selective gradients were created using Etest strips as described above and previously (5).Cells to be passaged were collected from the entire ZOI and a 1 cm region in the bacterial lawn surrounding the ZOI (Fig. 1) from the prior day's plate, suspended in TSB to a ~0.5 McFarland standard, and 200 µL spread onto a new GCB-K plate containing a fresh Etest strip.MICs were recorded each day of the experiment.Strains were exposed to azithromycin and penicillin for 20 days.Controls for each species were passaged on GCB-K plates as described above; however, they did not contain any antibiotic.
Doubling time was assessed for each strain used within the study.In brief, over night cultures on GCB-K media were inoculated into GCP broth supplemented with 1% Kelloggs solution to an OD 600 of ~0.1 (n = 4 replicate suspensions per species).OD 600 readings were taken using a Genesys 150 spectrophotometer (Thermo Scientific, Waltham, MA, USA) with measurements taken every 30 minutes for 24 hours with a 30-second shake at 180 cpm prior to reading.The BioTek Gen5 v.3.05 software was used for data interpretation and export.

Genomic sequencing and comparative genomics
DNA was isolated from cells using the PureLink Genomic DNA Mini kit (Thermo Fisher Corp., Waltham, MA, USA), following lysis in TE buffer [10 mM Tris (pH 8.0) and 10 mM EDTA] with 0.5 mg/mL lysozyme and 3 mg/mL proteinase K (Sigma-Aldrich Corp., St. Louis, MO, USA).The resultant genomic DNA was treated with RNase A and prepared for sequencing using the Nextera XT kit (Illumina Corp., San Diego, CA, USA).Libraries were uniquely dual-indexed and pooled and sequenced on the Illumina MiSeq platform at the Rochester Institute of Technology Genomics Core using V3 600 cycle cartridges (2 × 300 bp).The sequencing quality of each paired-end read library was assessed using FastQC v0.11.9 (89).Trimmomatic v0.39 (90) 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.Draft assemblies had been previously published for all strains (54), except for N. cinerea AR-0944.This assembly was constructed using SPAdes v.3.14.1 (91), and all assemblies were annotated with Bakta v.1.8.1 (92).Assembly quality was assessed using QUAST (http://cab.cc.spbu.ru/quast/).Trimmed reads were mapped back to draft assemblies using Bowtie2 v.2.2.4 (93) using the "end-to-end" and "very-sensitive" options, and Pilon v. 1.16 (94) was used to call variant sites.Data analysis and visualizations were conducted in R (95).

FIG 1 FIG 2
FIG1 Azithromycin and penicillin-mediated selection of four species of commensal Neisseria.(A) Four species with distinct genetic backgrounds were selected as unique starting points for in vitro evolution to two antimicrobials.Each experimental replicate and species/drug combination can be envisioned as an independent "roll of the dice, " in which new derived mutations and evolutionary trajectories may emerge.In brief, four experimental replicates were passaged for each species and drug combination for 20 days on selective gradients created with Etest strips.Cells for each passage were selected by sweeping the entire ZOI and a 1 cm band in the bacterial lawn surrounding the ZOI.(B) Overall, after 20 days, evolved azithromycin MICs tended to be higher than of penicillin MICs, with species also differing in their evolutionary trajectories toward elevated MICs within a drug class.

FIG 3 FIG 4
FIG 3 Evolved MICs and MIC log-fold change values separated by drug and species.(A) For azithromycin, N. subflava and N. cinerea had significantly higher MICs compared to N. elongata after selection (Tukey's HSD: P = 0.036 and P = 0.036, respectively).(B) Species were not significantly different between any contrast for penicillin.However, between species fold-change in MIC after evolution was significantly different for (C) four contrasts for azithromycin (Tukey's HSD: P < 0.05) and (D) three contrasts for penicillin (Tukey's HSD: P < 0.01).

FIG 5 7 TABLE 2
FIG5 Identity of derived mutations in CDSs for drug-selected lineages for (A) azithromycin and (B) penicillin.The percentage of reoccurring mutations within a given gene is displayed as a heatmap, with brighter blue coloration indicating the more frequent occurrence of a mutation within a CDS in replicate evolved lineages for each species.