Storage stability of non-encapsulated pneumococci in saliva is dependent on null-capsule clade, with strains carrying aliC and aliD showing a competitive disadvantage during culture enrichment

Storage stability of non-encapsulated pneumococci in saliva is dependent on null-capsule clade, with strains carrying aliC and aliD showing a competitive disadvantage during culture enrichment | 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 Storage stability of non-encapsulated pneumococci in saliva is dependent on null-capsule clade, with strains carrying aliC and aliD showing a competitive disadvantage during culture enrichment View ORCID Profile Claire S. Laxton , View ORCID Profile Orchid M. Allicock , View ORCID Profile Chikondi Peno , View ORCID Profile Tzu-Yi Lin , Alidia Koelewijn , View ORCID Profile Femke L. Toekiran , Luna Aguilar , View ORCID Profile Anna York , View ORCID Profile Anne L. Wyllie doi: https://doi.org/10.1101/2025.04.12.648276 Claire S. Laxton 1 Department of Epidemiology of Microbial Diseases, Yale School of Public Health , New Haven, CT, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Claire S. Laxton For correspondence: claire.laxton{at}yale.edu awyllie{at}gmail.com Orchid M. Allicock 1 Department of Epidemiology of Microbial Diseases, Yale School of Public Health , New Haven, CT, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Orchid M. Allicock Chikondi Peno 1 Department of Epidemiology of Microbial Diseases, Yale School of Public Health , New Haven, CT, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chikondi Peno Tzu-Yi Lin 1 Department of Epidemiology of Microbial Diseases, Yale School of Public Health , New Haven, CT, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tzu-Yi Lin Alidia Koelewijn 1 Department of Epidemiology of Microbial Diseases, Yale School of Public Health , New Haven, CT, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Femke L. Toekiran 1 Department of Epidemiology of Microbial Diseases, Yale School of Public Health , New Haven, CT, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Femke L. Toekiran Luna Aguilar 1 Department of Epidemiology of Microbial Diseases, Yale School of Public Health , New Haven, CT, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anna York 1 Department of Epidemiology of Microbial Diseases, Yale School of Public Health , New Haven, CT, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anna York Anne L. Wyllie 1 Department of Epidemiology of Microbial Diseases, Yale School of Public Health , New Haven, CT, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anne L. Wyllie For correspondence: claire.laxton{at}yale.edu awyllie{at}gmail.com Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Background Non-encapsulated Streptococcus pneumoniae (NESp) represent up to 19% of circulating pneumococci and exhibit high rates of antimicrobial resistance. Saliva is increasingly used as a pneumococcal carriage study specimen, and we recently developed a qPCR assay to enhance carriage surveillance and characterization of NESpn in saliva. Previous work has established that pneumococci remain viable in unsupplemented saliva for extended periods under various conditions, however these findings may not be applicable to NESp. Therefore, to ensure the robustness of NESp detection in saliva-based carriage studies we evaluated the impact of transport and storage conditions of saliva samples on NESp detection. Methods Six NESp strains from two clinically relevant NESp null capsule clades (NCC), NCC1 (carrying pspK ) and NCC2 (carrying aliC and aliD ), were spiked into lytA -negative saliva and incubated through various temperatures and freeze-thaw conditions. Endpoints were processed using either culture-enrichment and DNA extraction (CE-DNA), or an extraction-free method without CE, before testing for lytA using qPCR. Detection stability was assessed using regression modelling over temperature, time and freeze-thaws. Results Following CE-DNA, detection of NESp remained stable for ≤24 or ≤72 hours when stored at room temperature or 4°C, respectively, and over 2 freeze-thaw cycles (-80°C), with glycerol-supplementation providing slight benefits. Stability of detection when using CE-DNA depended on NCC; detection of NCC2 strains was lower, and less stable than NCC1. Compared to CE-DNA, extraction-free detection was more stable, with no significant loss over 72 hours at room temperature and over 3 freeze-thaw cycles. With extraction-free detection, there were also negligible diderences in detection between NCC1 and NCC2. Additionally, extraction-free detection of NCC1, and less so NCC2, increased over the first 24 hours when stored at 20-30°C, suggesting growth in saliva. Testing of ΔaliCaliD and ΔpspK mutants revealed these genes increased in vitro viability of NCC2 and NCC1, respectively, but did not significantly alter competitive fitness during CE. Conclusion NCC1 NESp strains exhibit similar stability patterns in unsupplemented saliva as encapsulated pneumococci. NCC2 strains, however, are less resilient during CE, likely due to competition with other oral microbes. Therefore, recovery of NCC2 NESp may be impacted by transport and storage conditions, leading to an underestimation of carriage prevalence when tested using CE-based methods. For the reliable carriage surveillance of NESp, samples should be stored at 4°C soon after collection and at -80°C within 72 hours. Methods which directly detect DNA without CE may provide a less biased accounting of NCC2 strains. Introduction The upper respiratory tract commensal bacterium, Streptococcus pneumoniae (pneumococcus) remains the leading global cause of morbidity and mortality from lower respiratory infections. The introduction of pneumococcal conjugate vaccines (PCVs) has led to a substantial reduction in pneumococcal disease caused by up to 21 of the 100+ known pneumococcal serotypes [ 1 , 2 ]. Concomitantly, PCV use has driven shifts in pneumococcal population dynamics, leading to increased prevalence of non-vaccine targeted serotypes (NVTs) and non-encapsulated strains [ 3 – 5 ]. Non-encapsulated Streptococcus pneumoniae (NESp) account for 3-19% of circulating pneumococcal strains and exhibit high rates of multi-drug resistance [ 6 ]. NESp are divided into Group I, which retain non-functional capsular genes, and Group II, which completely lack capsular genes in the cps locus [ 7 ]. Group II NESp are further classified into Null Capsule Clades (NCC) based on the presence of specific genetic elements within the cps locus, including pspK (NCC1), aliC and aliD (NCC2), only aliD (historically NCC3) or transposable elements alone (NCC4) [ 6 , 8 – 10 ]. While the absence of capsule generally reduces virulence, NCC1 and NCC2 strains have been associated with conjunctivitis outbreaks, otitis media and occasional cases of invasive pneumococcal diseases [ 6 , 11 – 15 ]. The genes present in NCC1 and NCC2 strains, pspK, aliC and aliD, are virulence factors which can compensate for the loss of capsule. AliC and AliD regulate the expression of choline-binding protein AC (CbpAC), which helps reduce C3b deposition on the bacterial surface, thereby enhancing resistance to classical complement-mediated clearance [ 16 , 17 ]. PspK increases biofilm formation and bacterial adherence to epithelial cells. In turn, this leads to enhanced transmission during influenza co-infection and reduces nasopharyngeal clearance due to interactions with secretory IgA [ 8 , 18 , 19 ]. Carriage prevalence of NESp is increasing, which is concerning due to their heightened propensity for genetic exchange, including antibiotic resistance elements [ 14 , 18 , 20 – 25 ]. Often, pneumococcal carriage studies neglect to further classify serologically non-typable strains, thus the prevalence of NCC1 and NCC2 NESp is most likely underreported [ 5 , 6 , 23 , 26 ]. We recently sought to remedy this by developing a multiplex-qPCR method to simplify the classification NCC1 and NCC2 and increase the inclusion of NESp monitoring in future carriage studies [ 27 ]. While nasopharyngeal swabs remain the gold-standard sample for pneumococcal carriage detection [ 28 ], swab collection is invasive and resource-intensive, requiring specialized materials and trained healthcare professionals. Saliva is a more accessible sample type than swabs [ 29 ] and is increasingly being used for pneumococcal carriage detection [ 30 – 35 ]. As such, the use of saliva for community-based studies continues to be validated and optimized. For example, we and others have demonstrated the high storage stability of respiratory pathogens, including encapsulated pneumococci, in unsupplemented saliva samples [ 36 – 39 ]. However, the stability of NESp, particularly the potentially more virulent NCC1 and NCC2 strains, has not yet been similarly investigated. Therefore, we examined the stability of the detection of NESp in saliva, over diderent temperatures, timespans and freeze-thaw cycles, to better inform the experimental design of future saliva-based pneumococcal carriage studies and improve the accuracy of NESp carriage estimates. Methods Saliva samples Participants were asked to provide whole-mouth unstimulated saliva by passively drooling saliva into a sterile 25 mL polypropylene tube at least 30 min after eating, drinking, or brushing their teeth. Samples were transferred at room temperature to the laboratory within 30 minutes of collection for temporary storage at 4°C, and aliquots were made and stored at -80°C within 12 h. Each sample was thoroughly screened for absence of pneumococci by incubating an aliquot of each sample at 37°C, and tested at 24, 48 and 72 h for the genes encoding the major pneumococcal autolysin LytA ( lytA) [ 40 ] and pneumococcal iron uptake ABC transporter lipoprotein PiaB ( piaB) [ 41 , 42 ], using methods described previously [ 27 ]. Saliva samples which tested negative for both piaB and lytA were thawed, pooled, aliquoted and either placed on ice for immediate use, or stored until needed at -80°C. Bacterial isolates Pneumococcal isolates ( Table 1 ) representing the major null-capsule clades (NCC1, 2a and 2b) were obtained from Lance Keller and Larry McDaniel (University of Mississippi Medical Centre, USA), Moon H Nahm (University of Alabama, USA) and Ron Dagan (Ben-Gurion University, Israel) [ 15 , 43 , 44 ]. View this table: View inline View popup Download powerpoint Table 1: Strains of Non-encapsulated Streptococcus pneumoniae used in this study. MLST: multi-locus sequence type, GII-NE Group II Non-Encapsulated, NCC: Null capsule clade, Isolates were plated as a lawn onto tryptic soy agar II (TSA II) plus 5% (v/v) defibrinated sheep blood (blood plates) and incubated at 37°C with 5% CO 2 overnight. The lawn was harvested into 1 mL brain heart infusion (BHI) medium using a cotton swab and used to inoculate 45 mL BHI medium. Cultures were grown at 37°C with 5% CO 2 to an optical density at 620 nm (OD 620 ) of ∼0.6 absorbance units. Cultures were harvested by centrifugation at 4,000 × g , and pellets were resuspended in 5 to 10 mL BHI medium supplemented with 10% (v/v) glycerol and stored at −80°C. The bacterial concentration (CFU/mL) of each sample were determined by colony counting of serially diluted samples cultured on blood plates and incubated at 37°C with 5% CO 2 overnight. Preliminary work from this study (data not shown), and other studies have found that due to aggregate formation, a phenomenon not observed with encapsulated pneumococcus, vigorous vortexing is required between serial dilution of NESp [ 46 ]. The recommended approach, to dilute into individual 1.5 mL microcentrifuge tubes with vortexing for 5-10 secs between dilutions, was adopted here to ensure accurate colony counting. Experimental design For each isolate listed in Table 1 , concentrated stocks were diluted in BHI then spiked into saliva at final concentrations of 10 3 and 10 4 CFU/mL (final concentration of BHI <3% v/v), as described previously [ 36 ]. Spiked saliva samples were incubated at 4°C, room temperature (∼20°C), and 30°C. At 24 h, 48 h, and 72 h, the samples were vortexed, and 100 μL was removed from each for culture enrichment (plated immediately) to test for strain viability as described previously [ 27 , 36 ]. A further 50 µL of each sample was aliquoted and stored at -80°C, for extraction-free pneumococcal detection [ 39 ]. To determine the edect of freeze-thawing on the stability of pneumococcal detection, saliva was spiked with pneumococci to final concentrations of 10 3 and 10 4 CFU/mL as above (unsupplemented) or supplemented with BHI with 50% glycerol (final concentration 30% BHI v/v and 15% glycerol v/v). Aliquots were stored for a minimum of 3 h at either −20°C or −80°C to allow complete freezing, before being thawed at room temperature. Once thawed, the samples were vortexed, and 100 μL was removed from each sample for culture enrichment before the remainder was returned to −20°C or −80°C. This process was repeated twice more. Where sample volume was insudicient for processing due to technical issues, the datapoint was excluded. To further examine the recovery of strains immediately following spiking, representative NCC1 (MNZ11) and NCC2 (MNZ41) strains were spiked at 15,000 CFU/mL into five diderent lytA and piaB negative saliva samples collected and prepared as above, in a matched pairs fashion. Stains were spiked into 1 mL BHI in the same way in triplicate. Following spiking, samples were vortexed for 10 s then plated for culture enrichment within 10 mins. Saliva sample processing by CE-DNA-extraction or extraction-free methods Culture-enriched saliva samples were thawed at room temperature and DNA was extracted from 200 μL of each sample using the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (MVP I) using a KingFisher Apex instrument (ThermoFisher Scientific), with modifications [ 35 ]. The method is referred to here as CE-DNA-extraction. Spiked saliva samples were additionally processed using an extraction-free method, without culture enrichment, as described previously [ 39 ]. This method involves a lysis step with proteinase K, heat inactivation at 95°C for 10 minutes and immediate transfer of the treated sample (which we will refer to as lysate) for pneumococcal detection via qPCR. Detection of pneumococcal carriage Each sample (2.5 µL of either DNA template or extraction-free lysate) was tested using the same dualplex qPCR targeting piaB and lytA as described above. Genomic DNA extracted from a pneumococcus serotype 19A strain ( lytA and piaB positive) was included in every plate as a positive control, using at least two standard concentrations (0.0001-1 ng/µL). Assays were run on a CFX96 Touch instrument (Bio-Rad) under the following conditions: 95°C for 3 min followed by 45 cycles of 98°C for 15 s and 60°C for 30 s. Samples were considered positive for pneumococci when the Cq values for both genes were ≤40 and within 2 Cq of each other and negative controls were undetectable. Since NESp strains MNZ41, C144.66 and D48 are naturally piaB negative, only lytA positivity was considered for samples spiked with these strains [ 27 ]. Plate-to-plate variation was corrected for by multiplying each sample Cq by an adjustment factor as described previously [ 27 ]. Statistical analyses Linear regression using R (version 3.5.2) was conducted to evaluate the impacts of time and temperature (time) or freeze-thaw cycle and supplementation (freeze thaw) on the detection of pneumococci from spiked saliva samples using the lytA adjusted Cq. Interaction terms were used to evaluate whether the edects of time and temperature (time), freeze-thaw cycle and supplementation (freeze thaw) and starting concentration (both) varied by strain. The ΔCq value represents the change in the mean Cq value from freshly spiked saliva under each condition (categorical). P values of less than 0.05 were considered significant. Tabulated model results are available in the Supplementary data. To further study viability following CE-DNA-extraction of NCC1 vs NCC2 strains immediately after spiking into saliva or BHI, a mixed-edects analysis was employed using GraphPad Prism (version 10.3.0) with Tukey’s multiple comparisons, utilizing a restricted maximum likelihood algorithm due to diderent sized groups. The experiment was conducted in a repeated measures fashion and the ΔCq value represents the change in the mean value between each strain. P values of less than 0.05 were considered significant. Results Detection of culture-enriched NESp depends on time, temperature, NCC and storage conditions When tested following CE-DNA-extraction, the stability of detection of NESp over time was dependent upon temperature ( Figure 1A ). There was no significant loss in detection over 72 h when stored at 4°C (ΔCq= 1.01, p= 0.083, 95% CI: -0.14 - 2.16). However, detection dropped significantly after 48 h when stored at RT (ΔCq= 3.44, p< 0.001, 95% CI: 1.50 - 5.38), and after 24 h when stored at 30°C (ΔCq= 4.86, p< 0.001, 95% CI: 2.76 - 6.97). In addition, stability of detection over time and temperature depended on NCC. When stratified by NCC, detection of NCC2 strains, but not NCC1, dropped significantly after 72 h when stored at 4°C (ΔCq= 1.68, p= 0.035, 95% CI: 0.12 – 3.24), after 48 h when stored at RT (ΔCq= 3.92, p= 0.006, 95% CI: 1.20 - 6.64), and for less than 24 h when stored at 30°C (ΔCq= 7.00, p< 0.001, 95% CI: 4.38 – 9.60). Download figure Open in new tab Figure 1: Detection of NCC1 NESp over time, temperature and freeze-thaw is more stable than that of NCC2 when spiked into saliva. Panel A and B show the detection of NESp over time at diFerent temperatures (4°C, RT= room temperature and 30°C) following either culture-enrichment and DNA extraction (CE + DNA) or extraction-free methods, respectively. Saliva was initially spiked at either 1,000 or 10,000 CFU/mL, as indicated by the right-hand label. Panel C and D show the detection of NESp over 3 freeze-thaw cycles following either CE + DNA or extraction-free methods, respectively. Saliva was initially spiked into unsupplemented saliva or saliva supplemented with BHI plus glycerol at either 1,000 or 10,000 CFU/mL and cycled through either -20°C or -80°C, as indicated by the right-hand label. Data are shown as the mean adjusted Cq for lytA for each of NCC1 (n=2, red) or NCC2 (n=4, blue) with the range shown as a shaded ribbon. We next assessed the edect of freeze-thaw cycles on detection stability of NESp ( Figure 1C ). For samples that were processed by CE-DNA-extraction, detection stability of NESp across freeze-thaw cycles was dependent on storage temperature. There was a significant reduction in detection by the second freeze-thaw cycle for samples stored at -20°C (ΔCq= 4.46, p< 0.001, 95% CI: 2.25 – 6.68) and by the third cycle for samples stored at -80°C (ΔCq= 2.34, p= 0.008, 95% CI: 2.25 – 6.68), suggesting a slightly greater freeze-thaw stability when stored at -80°C. When tested using CE-DNA-extraction, adding BHI+glycerol to the saliva significantly increased overall bacterial detection following freeze-thaws at -80°C (ΔCq unsupplemented vs glycerol= 3.79, p< 0.001, 95% CI: 2.58 – 5.00) and -20°C (ΔCq unsupplemented vs glycerol= 5.22, p< 0.001, 95% CI: 3.65 - 6.78), however glycerol supplementation did not adect the stability of detection through consecutive freeze-thaws (ΔCq 0 h vs 72 h= 1.62, p= 0.251, 95% CI: -1.16 – 4.41) ( Figure 1C ). Similarly, while overall detection of NCC2 was lower than that of NCC1 strains (ΔCq= 4.20, p< 0.001, 95% CI: 3.17 – 5.25), stability of detection across freeze thaw cycles was unadected by NCC strain (ΔCq= 0.42, p= 0.781, 95% CI: -2.56 – 3.40). Processing of NESp using an extraction-free method leads to greater stability of detection which is less dependent on storage conditions and NCC To better understand the impact of culture-enrichment on the detection of NESp strains which underwent the same storage conditions, spiked saliva samples were also tested using the extraction-free method, which does not include culture enrichment. Similarly to when tested using CE-DNA-extraction, detection was stable over 72 h when stored at 4°C (ΔCq= 0.50, p= 0.127, 95% CI: -0.15 – 1.15). However, when stored at room temperature there was a slight increase in overall detection at 24 h (ΔCq= -1.14, p= 0.017, 95% CI: -2.07 – -0.21), which became a decrease in detection by 72 h (ΔCq= 1.24, p= 0.10, 95% CI: 0.31 – 2.16), suggesting some strains may have grown a small amount in the saliva. At 30°C, overall detection dropped after 48 h (ΔCq= 4.91, p< 0.001, 95% CI: 2.86 – 6.97). Unlike with CE-DNA-extraction, there was no diderence in stability of detection of NCC at either 4°C or room temperature when processed using the extraction-free method ( Figure 1B ). Detection according to NCC only deviated when stored at 30°C, in which detection of NCC2 strains decreased significantly after 24 h (ΔCq= 2.88, p< 0.027, 95% CI: 0.35 – 5.40), whereas detection of NCC1 strains, like at room temperature, increased at 24 h (ΔCq= -2.93, p< 0.002, 95% CI: -4.55 – 1.30), before decreasing by 72 h (ΔCq= 1.92, p< 0.025, 95% CI: 0.29 – 3.54). This suggests that NCC1 strains, and less so NCC2 strains can grow in whole saliva at temperatures between 20-30°C. Detection of NESp remained stable over all three freeze-thaw cycles when processed using the extraction free method (ΔCq= 0.47, p= 0.058, 95% CI: -0.02 – 0.95), and we did not observe any impact on NESp detection over repeated freeze-thaw cycle by NCC strain (ΔCq= -0.14, p= 0.470, 95% CI: -0.51 – 0.23), or storage temperature (ΔCq= -0.12, p= 0.507, 95% CI: --0.23 – 0.47) ( Figure 1D ). Unexpectedly, addition of glycerol supplementation resulted in slightly lower overall detection when samples were processed using the extraction-free method (ΔCq unsupplemented vs supplemented= - 2.22, p< 0.001, 95% CI: 2.57 - 1.87). Glycerol is typically considered a qPCR enhancer and cryoprotectant [ 47 ], however it is possible that the addition of glycerol in this concentration or formulation reduced the ediciency of the proteinase K or heat lysis step, resulting in less DNA available for qPCR. Overall, there were twice as many samples in which lytA was not detectable at endpoint following CE-DNA-extraction (7.4%) than extraction-free processing (3.4%) ( Table 2 ). Almost all the samples in which lytA could not be detected (32/663) were originally spiked with an NCC2 strain, except for four unsupplemented saliva samples which were spiked with a NCC1 strain and underwent ≥2 freeze thaw cycles. View this table: View inline View popup Download powerpoint Table 2: Percentage of samples from which NESp was not detectable (ND) stratified by treatment group. Following the observation that NCC2 strains were less stable in saliva than NCC1 strains following culture enrichment, we sought to determine if this was driven by the presence of aliC and aliD . Strains belonging to NCC1 (MNZ11) and NCC2 (MNZ41) were spiked into saliva and BHI in parallel and immediately tested using the CE-DNA-extraction method. Recovery of viable NCC1 strains was significantly higher than NCC2 following spiking into saliva (mean ΔCq= -3.52, 95% CI: -4.67 – -2.37, p<0.0001; Figure 2A ). However, there was no significant diderence when spiked into BHI (mean ΔCq= - 0.06, 95% CI: -1.19 – 1.08, p=0.9972; Figure 2B ). Recovery of both strains was lower and more varied in saliva than in BHI, suggesting that a component of saliva has an impact on bacterial viability during culture enrichment, which was more pronounced for NCC2 strains. Download figure Open in new tab Figure 2: Recovery of NCC2 NESp is lower than NCC1 immediately after spiking into saliva, but not BHI, and this is not dependant on aliC or aliD. MNZ11 (red, filled circles) is NCC1 and MNZ41 (blue, filled circles) is NCC2, MNZ1131 (red, open circles) and JLB01 (blue, open circles) are pspK and aliC/aliD gene knockouts of MNZ11 and MNZ41, respectively. Strains were spiked at a final concentration of 15,000 CFU/mL into either BHI (n=6 for MNZ11 and MNZ41, n=3 for MNZ1131 and JLB01) or matched pairs of lytA and piaB negative saliva from healthy individuals (n=11 for MNZ11 and MNZ41, n=6 for MNZ1131 and JLB01). Line is the mean, significance is shown for multiple comparisons between MNZ11 vs MNZ41, MNZ11 vs MNZ1131 and MNZ41 vs JLB01, **p<0.005, ***p<0.0005, ****p<0.0001. Knockout mutants MNZ1131 ( ΔpspK ) and JLB01 ( ΔaliCaliD ) were also tested, and while recovery in BHI was significantly lower for both than their wildtypes ( Figure 2B ), there were no significant diderences in recovery in saliva. This suggests that while pspK and aliC and aliD do play a role in overall cell viability, they are not primarily driving the edect on viability of capsule loss during culture enrichment in saliva. Discussion Carriage studies are reporting general increases in the prevalence of NESp, which is of concern particularly in the case of NCC1 and NCC2 strains, due to the increased virulence of these strains, and their propensity to acquire AMR genes [ 5 , 6 , 23 – 25 ]. There has also been an increase in recent years in the use of saliva as a sample type for investigating pneumococcal carriage, however the impact of saliva sample storage on the detection and recovery of NESp has not yet been described [ 30 , 32 , 34 , 41 , 48 – 50 ]. Therefore, in this study we sought to build upon the existing evidence examining encapsulated pneumococci viability in saliva across a range of storage conditions and determine if similar findings applied to NESp. We previously demonstrated that the viability of pneumococci remained stable in self-collected, unsupplemented saliva samples stored at 4°C to 30°C for up to 24 hours, and a portion of viable pneumococci could be detected for up to 72°C, though at lower quantities [ 36 ]. Findings from this current study demonstrate that while NCC1 strains exhibit equivalent storage stability in saliva to encapsulated strains [ 36 ], NCC2 strains are less able to survive culture enrichment, leading to lower detection than NCC1 following CE-DNA-extraction ( Figure 1A and C , Figure 2A ). Overall, when tested following the extraction-free method, detection was generally lower compared with CE-DNA-extraction, which was expected, due to the lack of enrichment and DNA concentration steps. Interestingly though, compared with CE-DNA-extraction, the extraction-free method led to more stable detection of NESp over both time ( Figure 1B ) and freeze-thaw cycles ( Figure 1D ), with negligible variation between NCC1 and NCC2. There were also fewer endpoint samples with a total loss of detection when using the extraction-free method compared with CE-DNA-extraction (3.4% vs 7.4%, Table 2 ). Culture enrichment amplifies all viable gentamycin resistant strains in a sample, the totality of which is harvested for detection [ 30 , 41 ]. Thus, for pneumococcal detection using culture enrichment, any pneumococcal strains present must be both viable at the time of plating, and able to survive on the culture plate during incubation. In the case of saliva, culture enrichment can include numerous mitis-group streptococci, which compete for resources and space [ 51 ]. Meanwhile, the extraction-free method used in this study consists simply of heat and enzymatic lysis step and direct-qPCR, meaning bacteria in the sample do not need to be viable to be detected. Therefore, it was hypothesised that the observed reduction in recovery of NCC2 strains was due to reduced cell survival in saliva and/or growth during culture enrichment. When we compared the recovery of cells immediately after spiking into either saliva or BHI, we found that recovery of NCC2 strains was significantly lower than that of NCC1 strain when culture-enriched form saliva, but not BHI ( Figure 2 ). Our findings suggest that while all cells were viable at the time of plating, NCC2 strains, but not NCC1, had a growth disadvantage in saliva during culture-enrichment. Potential explanations for this include a lower competitive fitness of NCC2 against other gentamicin-resistant bacteria present in saliva, or a salivary factor, such as a protease, that specifically inhibits their growth. Further studies are needed which test the recovery of NCC1 and NCC2 strains from either heat-treated or filtered saliva, to systematically determine the salivary component driving reduced NCC2 fitness during CE. Nevertheless, these data suggest that while CE may enhance detection of encapsulated pneumococci and NCC1 NESp, it can cause undue suppression of NCC2 NESp detection and possibly contribute to underreporting of NESp prevalence. Given that the extraction-free method is cheaper and less laborious than CE-DNA-extraction [ 39 ], it may be advisable to employ this, or other culture-free methods to ensure accurate detection of NESp. This study examined stability at two diderent bacterial loads, 1,000 and 10,000 CFU/mL, and the observed stability trends were generally replicated between each, with the expected ∼3 Cq diderence ( Figure 1 ). This suggests that the detection and quantification methods employed here were robust. Moreover, the recovery of representative NCC1 and NCC2 strains spiked into BHI was both highly replicable and similar between strains ( Figure 2 ), suggesting that the observed variations between strains could not be explained by variations in the quantity or viability of bacteria being spiked into saliva. However, this study was limited by testing the stability of only a small number of NESp strains, and only in the Group II, NCC1 and 2 clades. Given the inter-strain diderences observed here, it would be interesting to assess Group I NESp stability, as knowledge of the impact of capsule loss on stability of pneumococci in saliva is essential for informing future carriage study sample collection and storage practices. NESp prevalence is likely underreported in carriage surveillance due to limited focus on the detection and characterisation of these strains. We recently sought to address this by developing a method to aid the detection of NESp, particularly in oral sample types [ 27 ]. To further address the knowledge gap around NESp carriage, this current study aimed to determine the conditions for saliva collection, storage and testing for optimal NESp detection. As such, we have demonstrated that NESp can be reliably detected in saliva for up to 72 hours when stored at 4°C, or 24 hours when stored at room temperature. We have also demonstrated that while glycerol supplementation slightly improves NESp detection when using culture enrichment, it is not otherwise necessary. We also showed that NCC1 strains exhibit comparable resilience to encapsulated pneumococci, while NCC2 strains appear to be at a competitive disadvantage during culture enrichment. Recoverability of viable NCC1 and NCC2 strains was not significantly related the presence of either pspK or aliC / aliD genes, respectively ( Figure 2 ). Further investigation is required to understand the mechanism underlying our observation of reduced NCC2 fitness in saliva during CE. These findings underscore the importance of refining detection methodologies and considering strain-specific diderences when designing future pneumococcal carriage studies. Author contributions Conceptualization: CSL, OMA, AY, ALW; Methodology: CSL, OMA, CP, ALW, AY; Investigation: CSL, OMA, CP, AMK, TL, FT, LA, AY; Formal Analysis and Visualization: CSL, CP; Supervision: CSL, AY, ALW; Writing – original draft: CSL, CP, OMA; Writing-review & editing: CSL, OMA, CP, AY, ALW. Conflict of interest and Funding disclosures ALW has received consulting and/or advisory board fees from Pfizer, Merck, Diasorin, PPS Health, Co-Diagnostics, and Global Diagnostic Systems for work unrelated to this project and was the Principal Investigator on research grants from Pfizer, Merck, and NIH RADx UP to Yale University and from NIH RADx, Balvi.io, and Shield T3 to SalivaDirect, Inc. ALW is currently employed by Pfizer, Inc. The authors have no conflicts of interest to declare in relation to this work. 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Share Storage stability of non-encapsulated pneumococci in saliva is dependent on null-capsule clade, with strains carrying aliC and aliD showing a competitive disadvantage during culture enrichment Claire S. Laxton , Orchid M. Allicock , Chikondi Peno , Tzu-Yi Lin , Alidia Koelewijn , Femke L. Toekiran , Luna Aguilar , Anna York , Anne L. Wyllie bioRxiv 2025.04.12.648276; doi: https://doi.org/10.1101/2025.04.12.648276 Share This Article: Copy Citation Tools Storage stability of non-encapsulated pneumococci in saliva is dependent on null-capsule clade, with strains carrying aliC and aliD showing a competitive disadvantage during culture enrichment Claire S. Laxton , Orchid M. Allicock , Chikondi Peno , Tzu-Yi Lin , Alidia Koelewijn , Femke L. Toekiran , Luna Aguilar , Anna York , Anne L. 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