Intra-host genomic variation of Haemophilus influenzae isolates from asymptomatic nasopharyngeal carriers involves genes encoding proteins with diverse inferred functions

Intra-host genomic variation of Haemophilus influenzae isolates from asymptomatic nasopharyngeal carriers involves genes encoding proteins with diverse inferred functions | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Intra-host genomic variation of Haemophilus influenzae isolates from asymptomatic nasopharyngeal carriers involves genes encoding proteins with diverse inferred functions Randall J. Olsen , View ORCID Profile S. Wesley Long , Yuvanesh Vedaraju , Sandra Tomasdottir , Helga Erlendsdottir , Ásgeir Haraldsson , Karl G. Kristinsson , James M. Musser , Gunnsteinn Haraldsson doi: https://doi.org/10.1101/2025.07.03.663008 Randall J. Olsen 1 Laboratory for Molecular and Translational Human Infectious Diseases Research, Center for Infectious Diseases, Houston Methodist Research Institute, and Department of Pathology and Genomic Medicine, Houston Methodist Hospital , Houston, Texas, USA 2 Departments of Pathology and Laboratory Medicine and Microbiology and Immunology, Weill Medical College of Cornell University , New York, New York, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: rjolsen{at}houstonmethodist.org S. Wesley Long 1 Laboratory for Molecular and Translational Human Infectious Diseases Research, Center for Infectious Diseases, Houston Methodist Research Institute, and Department of Pathology and Genomic Medicine, Houston Methodist Hospital , Houston, Texas, USA 2 Departments of Pathology and Laboratory Medicine and Microbiology and Immunology, Weill Medical College of Cornell University , New York, New York, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for S. Wesley Long Yuvanesh Vedaraju 1 Laboratory for Molecular and Translational Human Infectious Diseases Research, Center for Infectious Diseases, Houston Methodist Research Institute, and Department of Pathology and Genomic Medicine, Houston Methodist Hospital , Houston, Texas, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sandra Tomasdottir 3 Department of Clinical Microbiology, Landspítali - the National University Hospital of Iceland , Reykjavik, Iceland 4 Faculty of Medicine, School of Health Sciences, University of Iceland , Reykjavik, Iceland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Helga Erlendsdottir 3 Department of Clinical Microbiology, Landspítali - the National University Hospital of Iceland , Reykjavik, Iceland 4 Faculty of Medicine, School of Health Sciences, University of Iceland , Reykjavik, Iceland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ásgeir Haraldsson 4 Faculty of Medicine, School of Health Sciences, University of Iceland , Reykjavik, Iceland 5 Children’s Hospital, Landspítali - the National University Hospital of Iceland , Reykjavik, Iceland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Karl G. Kristinsson 3 Department of Clinical Microbiology, Landspítali - the National University Hospital of Iceland , Reykjavik, Iceland 4 Faculty of Medicine, School of Health Sciences, University of Iceland , Reykjavik, Iceland Find this author on Google Scholar Find this author on PubMed Search for this author on this site James M. Musser 1 Laboratory for Molecular and Translational Human Infectious Diseases Research, Center for Infectious Diseases, Houston Methodist Research Institute, and Department of Pathology and Genomic Medicine, Houston Methodist Hospital , Houston, Texas, USA 2 Departments of Pathology and Laboratory Medicine and Microbiology and Immunology, Weill Medical College of Cornell University , New York, New York, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gunnsteinn Haraldsson 3 Department of Clinical Microbiology, Landspítali - the National University Hospital of Iceland , Reykjavik, Iceland 4 Faculty of Medicine, School of Health Sciences, University of Iceland , Reykjavik, Iceland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Haemophilus influenzae is a human-specific pathogen that causes infections ranging in severity from otitis media to potentially fatal meningitis. It also asymptomatically colonizes the upper respiratory tract. Although intra-host genomic variation of H. influenzae has been investigated in some anatomic sites, the genes most frequently acquiring nonsynonymous (amino acid changing) or nonsense (protein truncating) single nucleotide polymorphisms (SNPs) during human carriage or infection remain largely unidentified. To study intra-host genomic variation of H. influenzae during human asymptomatic carriage in the nasopharynx, the genomes of 805 isolates recovered from 24 healthy Icelandic children were sequenced. Most children were colonized with isolates with a single multilocus sequence type (MLST), although some were concurrently colonized with isolates with multiple MLSTs. Intra-host genomic variation was discovered with 120 genes acquiring SNPs in at least one isolate. Among them, 69 genes were recurrently polymorphic in isolates recovered from multiple children, and 72 SNPs occurred in multiple isolates recovered from the same child. The polymorphic genes encode proteins with diverse inferred functions, including transcription regulators and putative virulence factors. Many of the proteins likely play roles in bacterial fitness, virulence, and host-pathogen molecular interactions. This intra-host variation study provides a model for understanding the genomic diversity acquired by H. influenzae during human asymptomatic carriage in the nasopharynx. INTRODUCTION Haemophilus influenzae , a human-specific pathogen, is a Gram-negative coccobacillus historically recognized as a cause of serious infections 1 , 2 . Strains are serotyped based on expression of capsular polysaccharides (types a–f) 2 . Before the introduction and broad distribution of a polysaccharide conjugate vaccine targeting H. influenzae type b (Hib), Hib was among the primary causes of life-threatening pediatric meningitis, bacteremia, pneumonia, and epiglottitis 3 , 4 . While infections caused by Hib and other encapsulated serotypes have markedly declined in populations with high vaccine uptake, unencapsulated (serologically nontypeable) strains have become more common 5 – 7 . H. influenzae (particularly unencapsulated strains) also commonly colonizes the upper respiratory tract of young children and elderly adults 2 , 8 – 11 . Although asymptomatic colonization rates vary by age, race, sex, season, country, and environmental exposures, they are particularly high among individuals with chronic respiratory tract conditions and close person-to-person contacts such as daycare center attendees 2 , 12 – 18 . Sequencing of bacterial genomes began in 1995 with H. influenzae strain Rd 19 . Since then, researchers have sequenced the genomes of more than seventeen thousand H. influenzae strains to study population genomic structure 20 – 24 . These studies have revealed new insights to genomic diversity, gene content, and gene polymorphisms, particularly among serologically nontypeable strains. However, intra-host genomic variation has been far-less studied, leaving many key questions unanswered 21 , 25 – 27 . Specifically, the genes that most frequently acquire nonsynonymous (amino acid-changing) or nonsense (protein-truncating) single nucleotide polymorphisms (SNPs) during human colonization or infection remain largely unidentified. Studies of intra-host genomic variation with influenza A virus 28 , SARS-CoV-2 28 , 29 , Klebsiella pneumoniae 30 , Streptococcus pyogenes 31 , and other human pathogens 32 – 37 have provided key insights to microbial pathogenesis, including host-pathogen molecular interactions, strain fitness and virulence. To address this gap for H. influenzae , we recently investigated 38 intra-host genomic variation in serologically nontypeable isolates obtained from ear drainage fluid collected from children with otitis media. The analysis demonstrated that intra-host genomic variation in the middle ear affects numerous genes encoding proteins with diverse inferred functions 38 . To expand on this line of investigation, we sequenced isolates recovered from nasopharyngeal swabs of healthy children with asymptomatic carriage. Materials and Methods Specimen collection and H. influenzae culture Since 2009, investigators at Landspítali - the National University Hospital of Iceland have conducted an annual surveillance study examining asymptomatic carriage of H. influenzae , Streptococcus pneumoniae , and S. pyogenes in the Reykjavik capital region 39 – 41 . In March of each year, researchers visit 15 daycare centers, which are randomly assigned alphabetic designations to preserve anonymity ( Table 1 ), located across the Reykjavík Capital Region, comprising Reykjavík and 6 neighboring municipalities. The Reykjavík Capital Region accounts for approximately 65% of the Icelandic population (01 May 2025, www.statice.is ) 42 . Parents provided informed consent for nasopharyngeal swab collection and completed a questionnaire on recent antibiotic use. Each year, one nasopharyngeal swab sample (eSwab, Copan Italia s.p.a., Brescia, Italy) is collected from approximately 500 healthy children aged 1 to 6 years for asymptomatic carriage analysis ( Figure 1 ). In 2017 and 2018, 507 and 467 children were sampled, respectively. Swabs were immersed in Amies transport medium, and within 18 hours, swabs were plated on one blood agar plate and one chocolate agar plate with a bacitracin disc (Becton, Dickinson, and Company, Vaud, Switzerland) to isolate H. influenzae . Swabs were also plated on MacConkey, tryptic soy blood, nutrient, and SS agars to recover S. pneumoniae and S. pyogenes isolates not used in this study. Plates were incubated aerobically in the presence of 5% CO 2 for 18–20 hours at 36°C. Within 6 hours of plating, swabs were frozen at −80°C without cryopreservative additives. H. influenzae was identified using its characteristic colony morphology, resistance to bacitracin, and proteome from MALDI-TOF mass spectrometry (Bruker Daltonics, Billerica, MA). Bacterial growth was semi-quantified on a scale of 0 to 3+ following standard clinical microbiology laboratory procedures 43 . Children with nasopharyngeal swabs producing 3+ H. influenzae colonies were randomly assigned a numeric designation for anonymity ( Table 1 ). Download figure Open in new tab Fig. 1 Specimen acquisition, processing and intra-host genomic variation analysis methods. View this table: View inline View popup Table 1. Demographic information for the 24 children. H. influenzae isolate recovery for intra-host variation analysis For this study, nasopharyngeal swabs from 24 children collected in 2018 that produced 3+ H. influenzae colonies were selected for intra-host genomic variation analysis ( Table 1 ). Two children (Child 27 and Child 29) had nasopharyngeal swabs available from 2017 that were also included. Swabs were removed from the freezer and plated on two chocolate agar plates that were incubated overnight at 36°C with 5% CO 2 ( Figure 1 ). All isolated colonies ( n =13 to 43 colonies per swab; n =805 colonies total) were picked (Table S1), systematically labeled, subcultured, and confirmed as H. influenzae by MALDI-TOF mass spectrometry. Subsequently, a loopful of colonial material from each subculture, representing one colony recovered from the nasopharyngeal swab, was cryopreserved to create a stock. The stocks were later plated, incubated, collected onto a transport swab submerged in charcoal media (Medical Wire and Equipment Co Ltd, U.K.), and transferred to the Houston Methodist Research Institute for whole-genome sequencing ( Figure 1 ). Whole-genome sequencing The genomes of the 805 H. influenzae isolates were sequenced with well-established protocols 38 , 44 – 47 . Briefly, Illumina short-read sequencing was performed on all isolates. Bioinformatic analysis determined the multilocus sequence type (MLST) of each isolate (described below). To generate one closed reference genome for isolates of each MLST from each child, Oxford Nanopore Technology long-read sequencing was also performed ( n =39 genomes, Table 2 ). For both sequencing technologies, isolates were incubated overnight at 37°C in the presence of 5% CO 2 on chocolate agar, with DNeasy Blood and Tissue kits (Qiagen, Germantown, MD) used for genomic DNA extraction. Nextera XT kits and a NextSeq 550 instrument (Illumina, San Diego, CA) were used for short-read sequencing. V14 Native Barcoding Kits, R10.4 flow cells, and a GridION instrument were used for long-read sequencing (Oxford Nanopore Technologies, OX4 4DQ, UK). View this table: View inline View popup Table 2. H. influenzae asymptomatic carriage reference genomes. Whole-genome sequence analysis Whole-genome sequence analysis was performed using established protocols 38 . Briefly, Trimmomatic, Musket, and Fastq-pair were used for quality trimming, adaptor decontamination, and read pairing 48 – 50 . SRST2 and custom database files were used to assess gene content, including alleles for multilocus sequence typing, capsule genotyping, and fucose operon genotyping 51 . SRST2 and PlasmidFinder.fasta and ARGannot.r1.fasta database files were used to identify plasmid replicons and antimicrobial resistance genes 51 . Bifrost and Corer were used to determine the H. influenzae core genome from 99 genome sequences (Table S2) deposited in the NCBI Microbial Genome Database (accessed 05/01/2025) 52 , 53 . MUMmer dna-diff was used to determine phylogenetic relationships among the 805 H. influenzae asymptomatic carriage isolates relative to the core genome 54 . Rapidnj, SplitsTree, and Dendroscope were used to generate and visualize phylogenetic trees 55 . Reference genome assembly, closure, and annotation Oxford nanopore long reads were generated for one isolate representing each Child-MLST group ( Table 2 ). FastQC was used to assess read length and sequence quality, and MinKNOW (v.24.11.8) with high accuracy parameters was used to call nucleotides (Oxford Nanopore Technologies). Reference genomes were hybrid-assembled from the long- and short-read sequences. Filtlong (v.0.2.1), applying a minimum read length of 1 kb length and a retention threshold of 90%, was used to filter the long-read FASTQ files 56 . Short-read FASTQ files were trimmed and filtered using Trimmomatic (v.0.39) 48 . Hybrid genome assembly was initially conducted with Unicycler using the “normal” setting (v.0.5.0), SPAdes (v.3.15.4), and Racon (v.1.5.0) 57 – 59 . Genomes that did not assemble to closure were reattempted using the Unicycler “bold” setting. Genomes that did not assemble with Unicycler were reassembled using Trycycler in combination with Minimap2 (v.2.17-r941), Miniasm (v.0.3-r179), Raven (v.1.8.1), Minipolish, Polypolish, and POLCA 46 . PROKKA with default settings was used to annotate the closed circularized reference genomes 60 . Artemis (v.18.2.0) was used for genome visualization 61 . Intra-host genomic variation determination Contigs were assembled from the trimmed, error-corrected, paired short-reads using SPAdes 57 . MUMmer dna-diff was used to determine polymorphisms relative to the Child-MLST-matched reference genome 54 . An exclusion file for each closed circularized reference genome was created by identifying mismapped short reads to itself. Genes encoding transfer RNAs, ribosomal proteins, and pilis proteins were excluded due to their highly repetitive coding regions. Hypothetical genes were also excluded since they have uncertain inferred functions. Prephix, Phrecon, pre2snpfx, and snpfx in-house scripts were used for polymorphism data processing as previously described 30 . MEGA (v.11) was used to determine pairwise distances between strains 62 . Rapidnj, SplitsTree, and Dendroscope were used to generate and visualize phylogenetic trees 55 . Annotated genes with nonsynonymous (amino acid-changing) or nonsense (protein-truncating) SNPs compared to the reference genome underwent manual curation. UniProt and literature review were used to assign an inferred function to the protein encoded by each polymorphic gene 63 . Prism (v.10.4.2) was used for graphing and statistical analysis (GraphPad Software, Boston, MA). RESULTS Gene content analysis To assess intra-host genomic variation during human asymptomatic carriage, whole-genome sequencing of 805 H. influenzae isolates recovered from nasopharyngeal swabs collected in 2018 from 24 healthy Icelandic children ( n =13 to 43 isolates per child) was performed ( Figure 1 and Table 1 ). Samples collected in 2017 were also available for two children (child 27 and child 29). First, to assess genetic relationships among the asymptomatic carriage isolates, MLSTs were determined. In total, 39 distinct child-MLST groups were identified ( Table 2 and S1). While 17 children were colonized with isolates with a single MLST, 7 children were concurrently colonized with isolates with multiple MLSTs. Child 27 and child 29 were colonized with isolates from multiple different MLSTs during 2017 and 2018 (Table S1). In most instances, isolates with different MLSTs differed at multiple non-contiguous loci, suggesting concurrent asymptomatic carriage of distantly related lineages (Table S2). Additionally, isolates from four MLSTs were carried by multiple children ( Table 2 , S1, and S2). The clonal complexes containing the 39 MLSTs are known causes of human infection 64 . Gene content across the 805 H. influenzae asymptomatic carriage isolates was analyzed (Table S1). First, the presence of the fucose ( fuc ) operon was assessed, because previous reports indicate that fucK deletion occurs in some H. influenzae isolates and may lead to an undefined MLST 65 , 66 . All isolates had an intact fuc operon, except MLST2956 isolates from child 15 (Table S1). Second, genes associated with capsule production, plasmid replication, and antimicrobial resistance were identified. All isolates were unencapsulated (nontypeable) and did not contain plasmid replicons (Table S1). Although most isolates lacked antimicrobial resistance genes, a TEM-1D beta-lactamase was present in isolates recovered from child 2 (1 of 1 MLST652 isolates and 12 of 13 MLST1040 isolates), child 27 (all 14 MLST683 isolates), and child 59 (3 of 11 MLST280 isolates and 1 of 21 MLST1409 isolates). Notably, parental surveys indicated no recent antibiotic treatment for the three children with isolates with a TEM-1D beta-lactamase. Phylogenetic analysis To place the 805 H. influenzae asymptomatic carrier isolates into a broader phylogenetic context, a phylogenetic tree using SNPs determined relative to the H. influenzae core genome was constructed (Table S3). This analysis revealed two key findings. First, the 39 child-MLST groups were genomically diverse, differing at 192,419 unique SNP loci, representing 10.3% of the nucleotides in the core genome ( Figure 2 ). Second, isolates with the same MLST from different children formed closely related yet distinct subpopulations. For example, isolates classified as MLST159 from child 7 and child 39 were closely aligned relative to isolates from other MLSTs ( Figure 2 ) but formed distinct subpopulations ( Figure 3 ). Subsequent intra-host variation analyses for isolates from each child-MLST group were performed independently due to this high genomic diversity and evidence of distinct molecular evolutionary pathways. Download figure Open in new tab Fig. 2 Phylogeny of H. influenzae isolates. Phylogeny was inferred by neighbor-joining based on SNPs identified by Illumina short-read whole-genome sequencing of the 39 closed circularized reference genomes with read mapping relative to the H. influenzae core genome. Isolates (A) MHI3391 and MHI3401, (B) MHI3511, (C) MHI3512, (D) MHI3543, (E) MHI3338, (F) MHI4050, (G) MHI3578 and MHI4160, (H) MHI3361, (I) MHI3976, (J) MHI4015, (K) MHI4227, (L) MHI4194, (M) MHI3301 and MHI3481, (N) MHI3421, (O) MHI4090, (P) MHI3652, (Q) MHI4264, (R) MHI3620, (S) MHI3655, (T) MHI3428 and MHI3685, (U) MHI4051, (V) MHI3941 and MHI3963, (W) MHI3546, (X) MHI4060, (Y) MHI3332 and MHI3716, (Z) MHI4262, (AA) MHI4297, (BB) MHI3331, (CC) MHI4332, (DD) MHI3341, (EE) MHI3542, (FF) MHI4125, and (GG) MHI3451 are shown. Genomically closely related isolates, such as the same MLST or a single locus variant, may appear as overlapping circles. Download figure Open in new tab Fig. 3 Phylogeny of MLST159 H. influenzae isolates recovered from child 7 and child 39. Phylogeny was inferred by neighbor-joining based on SNPs identified by Illumina short-read whole-genome sequencing with read mapping relative to the H. influenzae core genome. Intra-host genetic variation analysis To identify H. influenzae genes undergoing intra-host genomic variation within each child’s nasopharynx, a closed circularized reference genome for each child-MLST group was assembled ( Table 2 ). A comparable strategy has been effectively used in prior studies of closely related organisms 30 , 38 , 45 , 46 , including intra-host genomic variation analysis of H. influenzae isolates recovered from otitis media patients 38 . Using clonally related reference genomes allows for highly accurate SNP determination 67 . The resulting 39 closed circularized reference genomes ranged from 1,775,912 to 2,022,617 base pairs and included 1,768 to 2,079 genes ( Table 2 ), which is consistent with the genome sizes and gene counts of other publicly available H. influenzae genomes (Table S2). For each H. influenzae asymptomatic carrier isolate, SNPs were determined relative to the child-MLST-matched reference genome. A total of 120 genes (representing 6.3% of the mean 1,897 genes in the reference genomes) acquired nonsynonymous (amino acid-changing) or nonsense (protein-truncating) SNPs in at least one isolate (Table S4). No gene deletions or frameshift mutations were identified. Among the 120 polymorphic genes, 69 had recurrent polymorphisms across isolates from multiple children ( Table 3 ), and 72 SNPs appeared in multiple isolates from the same child (Table S4). The inferred functions of the proteins encoded by the polymorphic genes included amino acid metabolism ( n =3 genes), antibiotic synthesis ( n =1 gene), carbohydrate metabolism ( n =9 genes), cell wall biosynthesis ( n =19 genes), environmental stress response ( n =9 genes), glycolipid metabolism ( n =5 genes), iron metabolism ( n =5 genes), recombination and repair ( n =5 genes), transcriptional regulator ( n =8 genes), transcription and translation ( n =28 genes), small molecule transport ( n =26 genes), and virulence ( n =2 genes) ( Figure 4A ). Genes encoding proteins implicated in transcription and translation, small molecule transport, and cell wall biosynthesis were most frequently polymorphic across isolates from multiple children ( Figure 4B ). Additionally, these polymorphic genes were present in most of the 39 reference genomes (Table S5). Download figure Open in new tab Fig. 4 Function of genes undergoing intra-host genomic variation during human asymptomatic carriage. ( A ) Inferred function of the proteins encoded by the 120 genes that acquired a nonsynonymous or nonsense SNP in at least one asymptomatic carrier isolate. ( B ) Inferred function of the proteins encoded by the 69 genes identified as recurrently polymorphic in isolates recovered from multiple child-MLST groups. View this table: View inline View popup Table 3. Polymorphic genes in isolates recovered from children with asymptomatic carriage. Diversifying selection of recurrently polymorphic genes To test the hypothesis that amino acid substitutions and protein truncations identified in the 69 recurrently polymorphic genes occurred through diversifying selection, the nucleotide changes defining the 461 variant alleles were evaluated (Table S6). The nucleotide changes were significantly concentrated at the first two positions of variant codons, allowing rejection of the hypothesis of selective neutrality (378 observed vs. 307 expected if evenly distributed across all three positions; χ test, P < .0001, χ = 27.95, df = 1, z = 5.286). Further, the dN/dS ratio for the 69 recurrently polymorphic genes was 2.25, indicating positive selection for genomic diversification (Table S6). Taken together, these data support a model in which intra-host variation drives the emergence of new alleles through diversifying selection. DISCUSSION Asymptomatic carriage is a common biological phenomenon that may drive microbial genomic adaption and facilitate person-to-person transmission of many pathogens 68 . To investigate intra-host genomic variation of H. influenzae during asymptomatic carriage in the human nasopharynx, the genomes of 805 isolates recovered from 24 healthy Icelandic children were sequenced. Substantial intra-host genomic variation was discovered among many genes encoding proteins that likely contribute to strain fitness, virulence, and host-pathogen molecular interactions. Two key discoveries emerged from this analysis. First, the level of genomic diversification was high compared to other studies of intra-host variation. Specifically, 120 genes, representing 6.3% of the genome, acquired nonsynonymous or nonsense SNPs in at least one isolate. In total, 335 isolates (41.6%) acquired at least one SNP. For comparison, in our recent study of intra-host genomic variation in H. influenzae isolates from children with otitis media, only 88 genes (4.6%) in 56 isolates (11.2%) acquired SNPs 38 . Similarly, a previous intra-host genomic variation study of S. pyogenes in a nonhuman primate model of necrotizing myositis revealed SNPs in only 20% of isolates, with approximately 50% occurring in one gene 31 . Second, frequent recurrence of polymorphic genes in the nasopharyngeal isolates was observed. Of the genes with SNPs, 69 (57.5%) were polymorphic in isolates from multiple child-MLST groups, while 72 (60.0%) were polymorphic in multiple isolates within the same child-MLST group. That is, compared to other investigations of intra-host genomic variation, diversification of H. influenzae in human nasopharyngeal asymptomatic carriage was common. We recently investigated intra-host genomic variation in H. influenzae isolates recovered from Icelandic children with symptomatic otitis media 38 . Since the nasopharynx and middle ear have distinct anatomic and physiologic features that likely exert unique selective pressures, we hypothesized that different genes would undergo intra-host genomic variation at each site. Consistent with this hypothesis, only three genes were recurrently polymorphic in isolates from both sites: nanK (N-acetylmannosamine kinase NanK), patB (cystathionine beta-lyase PatB), and phoR (phosphate regulon sensor protein PhoR). Notably, phoR was the most frequently polymorphic gene (4/13, 30.8% of child-MLST groups) in isolates associated with otitis media and the third most polymorphic gene (13/39, 33.3% of child-MLST groups) in isolates from asymptomatic carriage 38 . Although the function of PhoR has not been studied in H. influenzae , in other bacterial species, PhoR serves as the signaling histidine kinase in a two-component regulatory system responsive to fluctuating phosphate levels 69 . Since phosphate is naturally present in body fluids, including those in the middle ear and nasopharynx, its levels may change in various disease and health states 70 , 71 . Phosphate acquisition from the host environment is essential for many human pathogens, including H. influenzae 72 . We hypothesize that phenotypic diversity in PhoR, arising through intra-host genomic variation, enhances fitness by enabling adaptation to the nutrient-limited environments of the middle ear and nasopharynx. PhoR also regulates the expression of many genes encoding proteins implicated in environmental stress response, serum bactericidal activity resistance, antimicrobial peptide resistance, and other functions that may further contribute to fitness 72 . As an additional example illustrating differing host-pathogen molecular interactions and selective pressures driving genomic diversification across different anatomic sites, our study of H. influenzae otitis media isolates revealed no acquired SNPs in genes encoding transcriptional regulators or putative virulence factors 38 . In comparison, in the current asymptomatic carrier study, eight transcriptional regulators and two putative virulence factors exhibited polymorphisms. Notably, both of the identified virulence factors target immunoglobin A (IgA), which is present in high concentrations in saliva and nasal secretions and acts as a key host immune defense molecule in the human upper respiratory tract 73 . In particular, esiB , which encodes a putative IgA-binding protein, acquired 31 distinct nonsynonymous or nonsense SNPs in isolates from eight child-MLST groups. Although esiB has not been previously studied in H. influenzae , other IgA-binding proteins have been recognized as critical virulence factors in many upper respiratory tract pathogens, such as S. aureus and S. pyogenes 74 – 77 . Similarly, iga , encoding the immunoglobin A1 protease autotransporter that cleaves IgA1 into Fc and Fab fragments, acquired three nonsynonymous SNPs within isolates from one child 78 . The proximity of these amino acid substitutions may represent a recombination event in this naturally competent pathogen. In general, IgA proteases are proven virulence factors 79 , 80 . In H. influenzae , IgA1 protease activity is significantly lower in asymptomatic nasopharyngeal carriage relative to sterile site infections 78 . Also, amino acid substitutions are known to be structurally destabilizing and reduce IgA1 protease activity 81 . The three amino acid substitutions occurred in the autotransporter domain, which could interfere with normal transport of the protease to the cell surface and reduce IgA1 protease activity 63 . We hypothesize that intra-host genomic diversification of esiB and iga may favor asymptomatic carriage by altering the ability of IgA to interact with bacterial antigens or host cell receptors to mute the immune response 82 . This asymptomatic carrier study is the largest H. influenzae intra-host genomic variation investigation to date, including the largest number of subjects and isolates per subject. However, there are some limitations to the study design. First, because the children naturally acquired H. influenzae , the exact duration of colonization remains unknown, which could introduce an unknown bias related to gene selection or SNP accumulation. Second, only a single sampling timepoint was obtained, so day-to-day or longitudinal fluctuations in bacterial burden are unknown. Third, the samples underwent multiple passages from swab collection to genome sequencing, which could have an unknown effect on isolate recovery. Fourth, selecting samples from children with the highest nasopharyngeal colonization density could also introduce biases related to SNP diversity. To summarize, whole-genome sequence analysis of 805 H. influenzae isolates from 24 healthy Icelandic children with asymptomatic nasopharyngeal carriage identified 120 polymorphic genes, including 69 genes that were recurrently polymorphic in multiple children. The genes encode proteins with diverse inferred functions that likely contribute to strain fitness, virulence, and host-pathogen molecular interactions. These data will generate new hypotheses bearing on novel targets for diagnostics, therapies, and vaccines. Future studies examining H. influenzae intra-host genomic variation with isolates from different anatomic sites or patient populations are warranted to expand on these findings. ETHICAL STATEMENT The study was approved by the Icelandic National Bioethics Committee (license no: 12.010.S1). FUNDING STATEMENT Research in the laboratory of JMM is supported in part by the Fondren Foundation. The study was partly funded in part by the Icelandic Research Fund grant no. 152047-051 and the Landspítali University Hospital Research Fund. CONFLICT OF INTEREST None declared. AUTHORS CONTRIBUTIONS Conceptualization: RJO, KGK, HE, AH, JMM, and GH. Specimen collection: ST. Strain culturing, banking, cataloging, and shipping: HE, and ST. Sequencing, data collection, sequence data analysis: RJO, SWL, and YV. Analysis: all authors. Original draft preparation RJO and GH. Writing, reviewing, and editing: all authors. All authors read, provided input, and approved the final manuscript. DATA AVAILABILITY Whole genome sequencing data for this investigation were submitted to the National Center for Biotechnology Information Sequence Read Archive and closed genomes were submitted to GenBank under BioProject accession PRJNA1248003. ACKNOWLEDGMENTS We thank the staff at the clinical diagnostic laboratory at Landspítali. 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