Inhibitory effect of Dolosigranulum pigrum and Corynebacterium pseudodiphtheriticum on pneumococcal in vitro growth

Inhibitory effect of Dolosigranulum pigrum and Corynebacterium pseudodiphtheriticum on pneumococcal in vitro growth | 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 Inhibitory effect of Dolosigranulum pigrum and Corynebacterium pseudodiphtheriticum on pneumococcal in vitro growth View ORCID Profile M Cisneros , M Blanco-Fuertes , A Lluansí , P Brotons , D Henares , A Pérez-Argüello , G González-Comino , P Ciruela , A Mira , View ORCID Profile C Muñoz-Almagro doi: https://doi.org/10.1101/2025.01.10.632320 M Cisneros 1 Grup de Recerca, Institut de Recerca Sant Joan de Déu (IRSJD), Hospital Sant Joan de Déu , 08950. Esplugues, Barcelona, Spain 3 Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya , Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for M Cisneros M Blanco-Fuertes 1 Grup de Recerca, Institut de Recerca Sant Joan de Déu (IRSJD), Hospital Sant Joan de Déu , 08950. Esplugues, Barcelona, Spain 2 CIBER de Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III , Madrid, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: carmen.munoza{at}sjd.es miguel.blanco{at}sjd.es A Lluansí 1 Grup de Recerca, Institut de Recerca Sant Joan de Déu (IRSJD), Hospital Sant Joan de Déu , 08950. Esplugues, Barcelona, Spain 2 CIBER de Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III , Madrid, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site P Brotons 1 Grup de Recerca, Institut de Recerca Sant Joan de Déu (IRSJD), Hospital Sant Joan de Déu , 08950. Esplugues, Barcelona, Spain 2 CIBER de Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III , Madrid, Spain 3 Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya , Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site D Henares 1 Grup de Recerca, Institut de Recerca Sant Joan de Déu (IRSJD), Hospital Sant Joan de Déu , 08950. Esplugues, Barcelona, Spain 2 CIBER de Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III , Madrid, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site A Pérez-Argüello 1 Grup de Recerca, Institut de Recerca Sant Joan de Déu (IRSJD), Hospital Sant Joan de Déu , 08950. Esplugues, Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site G González-Comino 1 Grup de Recerca, Institut de Recerca Sant Joan de Déu (IRSJD), Hospital Sant Joan de Déu , 08950. Esplugues, Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site P Ciruela 2 CIBER de Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III , Madrid, Spain 4 Agència de Salut Pública de Catalunya , Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site A Mira 2 CIBER de Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III , Madrid, Spain 5 Genomics & Health Department, FISABIO Foundation , Valencia, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site C Muñoz-Almagro 1 Grup de Recerca, Institut de Recerca Sant Joan de Déu (IRSJD), Hospital Sant Joan de Déu , 08950. Esplugues, Barcelona, Spain 2 CIBER de Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III , Madrid, Spain 3 Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya , Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for C Muñoz-Almagro For correspondence: carmen.munoza{at}sjd.es miguel.blanco{at}sjd.es Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Background Streptococcus pneumoniae is a nasopharynx coloniser that can invade sterile tissues, causing Invasive Pneumococcal Disease (IPD). Dolosigranulum pigrum and Corynebacterium pseudodiphtheriticum are commensal bacteria commonly isolated from the nasopharynx of healthy children, potentially playing a protective role. This study aims to analyse the effects of D. pigrum and C. pseudodiphtheriticum on S. pneumoniae in vitro growth. Methods Pneumococcal strains were collected from IPD patients and healthy carriers in Catalonia (2016-2023). D. pigrum and C. pseudodiphtheriticum strains were isolated from a healthy child’s nasopharynx. S. pneumoniae was co-cultured with each commensal bacterium in triplicate experiments. Pneumococcal growth was quantified using a real-time PCR assay targeting the lytA gene. The effect of commensal bacteria on pneumococcal growth was evaluated using a linear mixed-effect regression model. Results Twenty-eight pneumococcal strains expressing 24 different serotypes and 26 clonal types were analysed (18 isolated in blood and 10 in nasopharyngeal aspirate). Pneumococcal growth was decreased by D. pigrum (β = −0.763, 95% CI: −0.94 to −0.59, p < 0.0001) and C. pseudodiphtheriticum (β = −0.583, 95% CI: −0.76 to −0.41, p < 0.0001). The combined presence of both had a stronger inhibitory effect (β = −0.971, 95% CI: −1.15 to −0.79, p < 0.0001). No association was found between isolation site or serotype with pneumococcal growth. Conclusion D. pigrum and C. pseudodiphtheriticum significantly reduced pneumococcal growth, with a synergic effect when combined. This antagonistic effect supports the potential protective factor of healthy nasopharyngeal microbiota against IPD and the development of these microorganisms as probiotics. INTRODUCTION Streptococcus pneumoniae is a gram-positive bacterium that asymptomatically colonises the human nasopharynx, especially among children aged under 5 years. However, under certain circumstances, it has the potential to invade sterile tissues and cause disease ( 1 ). Invasive Pneumococcal Disease (IPD) is the most severe form of pneumococcal infection and includes bacteremia, pneumonia, meningitis, and septicemia, among others. Despite the availability of pneumococcal vaccines, it has been estimated that S. pneumoniae is responsible for over 300,000 deaths annually in children under five years of age ( 2 ). Pneumococcal conjugate vaccines (PCVs) are developed against the bacterium’s main virulence factor, the capsular polysaccharide (CPS) ( 3 ). There are more than 100 known capsular serotypes with different degrees of invasiveness; some of them are high invasive disease potential serotypes ( 4 – 6 ). Following the introduction of conjugate vaccines, numerous studies have shown a decreased prevalence of vaccine serotypes (VTs) and a significant reduction in IPD caused by these serotypes. Nevertheless, vaccines do not cover all serotypes, leading to the phenomenon of serotype replacement, in which non-vaccine serotypes (NVTs) are expanded and become more in disease ( 7 , 8 ). Pneumococcal nasopharyngeal colonisation represents a key factor to understand the burden of pneumococcal disease and address its prevention. Colonisation is a complex and dynamic process in which multiple factors interplay, including environmental, host, and microbiota factors. Intricate interactions with other common inhabitants of the nasopharynx seem to be an important step for pneumococcal colonisation ( 1 , 9 ). Previous studies have suggested that certain natural colonisers of the nasopharynx could play a protective role against the colonisation of S. pneumoniae ( 10 ). Significant abundances of Dolosigranulum pigrum and Corynebacterium species have been found in nasal and nasopharyngeal microbiota of children when S. pneumoniae is absent ( 10 – 12 ). Furthermore, increased abundances have been detected in healthy controls compared to patients with different respiratory infections, suggesting a potential beneficial role in respiratory health through negative interactions with common pathobionts ( 10 – 19 ). D. pigrum is one of the known species of the genus Dolosigranulum that was first reported ( 20 ) ( 21 ). In terms of structure, it is a gram-positive coccus arranged in pairs, tetrads, and clusters. This catalase-negative bacterium is generally sensitive to antibiotics like beta-lactams ( 22 ). A notable characteristic of D. pigrum that enhances it as a potential protective species is its ability to produce lactate, classifying it as lactic acid bacterium (LAB) ( 23 , 24 ). Additionally, genome sequencing has confirmed this classification by identifying genes associated with the homofermentation of carbohydrates ( 24 , 25 ). Most LABs have been found in the digestive tract, where they perform beneficial tasks for human health, such as enhancing resistance to pathogens through microbe-microbe interactions, the synthesis of bacteriocins, or immunomodulation ( 26 ). For instance, Lactobacillus murinus a LAB from the lung microbiota in mice can inhibit S. pneumonia growth in vitro and prevent colonization in vivo ( 27 ). D. pigrum is commonly isolated from the nasal cavity or the nasopharynx, where it coexists with other commensal bacteria such as Corynebacterium spp ( 11 ). It has been also described that Corynebacterium pseudodiphtheriticum , one of the species of this genus, could also prevent pathogenic bacteria colonisation in the nasopharynx in cooperation with D. pigrum ( 28 ). Brugger S, et al. demonstrated increased relative abundances of C . pseudodiphtheriticum were increased in the presence of D. pigrum . Additionally, they observed that C . pseudodiphtheriticum could enhance the growth of D. pigrum in vitro ( 14 ). Despite these suggestive results, little is known about the impact of D. pigrum and C. pseudodiphtheriticum on pneumococcal replication rate and the consistency of these results according to different capsular serotypes of S. pneumoniae . Thus, the aim of the present study is to analyse the effects of the two commensal bacteria, D. pigrum and C. pseudodiphtheriticum on Streptococcus pneumoniae in vitro growth (IVG) in a diverse collection of S. pneumoniae strains. MATERIAL AND METHODS Bacterial strains collections Invasive pneumococcal strains were obtained from the collection of the Support Laboratory for Molecular Epidemiology of IPD in Catalonia during 2016-2023. Carriage strains ( D. pigrum, C. pseudodiphtheriticum, and S. pneumoniae ) were prospectively collected with informed consent in the framework of two consecutive funded projects focused on the role of nasopharyngeal microbiota in respiratory health and disease during 2016-2023. All the strains were preserved in 1 ml of preservation media of skim milk at −80°C.The selected strains represented the main serotypes and clones detected in Catalonia during the study period as reported on Microbiological notification system of Catalonia. Isolation and identification process of Dolosigranulum pigrum and Corynebacterium pseudodiphtheriticum D. pigrum and C. pseudodiphtheriticum strains were isolated from a nasopharyngeal aspirate (NPA) sample of a healthy child attended at Sant Joan de Déu Barcelona Children’s Hospital (SJD) in the autumn of 2018. Isolation of both bacteria was performed by culturing 100 µl of NPA sample on Blood Agar plates (Columbia agar supplemented with 5% sheep blood; BioMérieux). Additionally, D. pigrum was isolated on CNA Agar plates (Columbia CNA agar supplemented with 5% sheep blood; Becton Dickinson) and Mannitol Salt Agar (Becton Dickinson). The plates were incubated at 37 °C under aerobic conditions supplemented with 5% CO 2 . MALDI-TOF mass spectrometry was used to identify both commensal bacteria. This system generates a spectrum based on the mass-charge relationship of the microorganism’s proteins, which is then compared with reference libraries containing the different spectra of known microorganisms ( 29 ). Isolation, identification, and molecular characterisation process of Streptococcus pneumoniae Pneumococcal isolates were obtained from blood and nasopharyngeal samples from IPD patients and healthy carriers in Catalonia during 2016–2023. To isolate S. pneumoniae, 100 µl of the NPAs were directly cultured in Blood Agar plates (Columbia agar supplemented with 5% sheep blood; BioMérieux) and incubated at 37 °C under aerobic conditions supplemented with 5% CO 2 . Pneumococcal identification was conducted using standard microbiological techniques, such as the optochin sensitivity test and colony morphology ( 30 ). In addition, capsular typing was performed on all pneumococcal using fluorescence fragment analysis and Whole Genome Sequencing (WGS) ( 31 , 32 ). According to previous literature, ( 5 ) Serotypes 1, 3, 4, 5, 7F, 8, 9A, 9V, 12F, 14, 18, 19A, 24F, and 33F were considered high invasive disease potential serotypes (HIPST) while the rest were considered low invasive disease potential serotypes (LIPST). In vitro growth of pneumococcal isolates according to D. pigrum and C. pseudodiphtheriticum exposure Preparation and standardisation of D. pigrum and C. pseudodiphtheriticum enriched media Isolates of D. pigrum and C. pseudodiphtheriticum preserved at −80°C were cultured on Blood Agar plates (Columbia agar supplemented with 5% sheep blood; BioMérieux). To establish different in vitro conditions for S. pneumoniae , overnight cultures of D. pigrum and C. pseudodiphtheriticum (grown in aerobic conditions with 5% CO 2 at 37 °C) were suspended in 2 ml of fresh BBL Todd-Hewitt broth (Becton Dickinson). The suspension solution was adjusted to a concentration of 6 log 10 genome copies per microlitre ± 0.7 log 10 , determined by Qubit Fluorometric Quantification from the extraction of genomic DNA ( Figure 1 ) . Download figure Open in new tab Figure 1: Flow diagram of the protocol for in vitro growth of pneumococcal isolates according to D. pigrum and C. pseudodiphtheriticum exposure In Vitro Exposure of S. pneumoniae to Commensal Bacteria-Enriched Media In order to expose S. pneumoniae to commensal bacteria, 1 CFU of each pneumococcal isolate was inoculated into four culture conditions, resulting in the following pneumococcal broth cultures: (i) S. pneumoniae cultured alone in 2 ml of BBL Todd-Hewitt broth (Becton Dickinson) as negative controls (S); (ii) S. pneumoniae co-cultured with D. pigrum (SD); (iii) S. pneumoniae co-cultured with C. pseudodiphtheriticum (SC); and (iv) S. pneumoniae co-cultured with both (SDC). Cultures were incubated under aerobic conditions with 5% CO 2 at 37 °C for 18 hours. To account for the intrinsic heterogeneity among strains, each strain was tested in triplicate ( Figure 1 ) . Quantitative Assessment of S. pneumoniae Growth DNA was extracted from 400 µl of each of the four pneumococcal cultures using the eMAG platform (bioMerieux; Marcy-l’Étoile, France) with a final elution volume of 50 µl. S. pneumoniae was quantified using a real-time PCR assay targeting the lytA gene following serial dilutions of 1:10 of the genomic extract. Primers and probes were employed according to the Centers for Disease Control and Prevention (CDC) guidelines ( 33 ). A calibration curve was established to correlate S. pneumoniae DNA concentration (ng/µl) with the Cycle Threshold (CT) values obtained from the real-time PCR. The calibration curves were generated from the extraction of genomic DNA from a Todd-Hewitt suspension of S. pneumoniae of each strain. The DNA concentration was measured using Qubit Fluorometric Quantification with serial 1:10 dilutions prepared from the range of 10 to 10 -6 nanograms per microliter (ng/µl). The resulting pneumococcal IVG values, initially quantified in ng/ µl, were converted to gc/µl using the median genome size of S. pneumoniae , which is 2.085 kbp ( 34 ) ( Figure 1 ) . Statistical analyses The effect of commensal bacteria on pneumococcal growth was considered according to microbial and clinical characteristics of pneumococcal infection: serotype invasiveness, healthy pneumococcal nasopharyngeal carriage or IPD status, and isolation site (blood or nasopharynx). A linear mixed-effect regression model (LMM) was used to evaluate the impact of D. pigrum and C. pseudodiphtheriticum on S. pneumoniae ’s growth. To normalise the distribution, the outcome variable, pneumococcal growth, underwent a log transformation. The strain identifier and the strain replicate were used as nested random effects. This approach allows for control and measurement of the inherent differences in pneumococcal growth between strains and ensures not biased results derived from fixed effects. All non-collinear variables examined—the culture condition, serotype invasiveness, and site of isolation—were included in an initial LMM. To determine which variables should be used as a fixed effect, a stepwise approach from the initial LMM was used to iteratively remove variables, resulting in different models. The Akaike Information Criteria (AIC), Restricted Maximum Likelihood (REML), and Determination Coefficient (R²) ( 35 – 37 ) were the three key adjustment criteria used in each step to assess model fit and identify the significant predictor variables. This process led to the selection of the optimal LMM, the LMM2. To assess the collinearity among the fixed effect variables, the Variance Inflation Factor (VIF) was used, and those with a VIF > 5 were deemed collinear ( 38 ). Associations between these variables were measured by Estimated coefficient (β), and their significance was assessed by estimated Marginal Means. 95% confidence intervals (CIs) were calculated, and statistical significance was determined by setting a p-value of 0.05. All the statistical analyses were performed using the 4.3.1 version of R and RStudio software ( 39 ) using the car ( 40 ), lme4 ( 41 ), emmeans ( 42 ), lmerTest ( 43 ), and MumIn ( 44 ) packages. RESULTS Pneumococcal strain’s features During the study period (2016-2023), a total of 28 S. pneumoniae strains were selected for analysis; 18 were isolated from blood and 10 from nasopharyngeal samples (2 from IPD patients and 8 from healthy children). Twenty-four different serotypes and 27 different clones were detected in the 28 pneumococcal strains. A proportion of 60.71% of serotypes (n=17) were classified as low invasive disease potential serotypes ( Table 1 ) . View this table: View inline View popup Download powerpoint Table 1: Characteristics of Pneumococcal strains included in this study Descriptive results All 28 pneumococcal strains were replicated three times, resulting in a total of 84 observations. Mean growth of pneumococcal isolates in monoculture (3.04 log 10 gc/µl; SD, 0.42) was higher compared to those co-cultured with D. pigrum (2.28 log 10 gc/µl; SD, 0.57), C. pseudodiphtheriticum (2.46 log 10 gc/µl; SD, 0.82), or both commensal species (2.07 log 10 cg/µl; SD, 0.78) ( Table 2 ). The mean growth of high invasive disease potential serotypes (2.61 log 10 gc/µl; SD, 0.75) pneumococcal isolates was higher than low invasive disease potential serotypes (2.35 log 10 gc/µl; SD, 0.74). Similarly, pneumococcal isolates from blood samples showed a higher mean growth (2.53 log 10 gc/µl; SD, 0.72) compared to those from NPA (2.32 log 10 gc/µl; SD, 0.79) ( Table 2 ) . View this table: View inline View popup Download powerpoint Table 2: Mean bacterial culture growth of 28 pneumococcal strains according to different categorical variables under various culture conditions Selection of optimal model Three LMMs were conducted to identify which predictor variables of S. pneumoniae strains had an effect on their growth. LMM2 presented lower AIC (580.748) and REML (564.7) values, indicating better fit compared to LMM1 and LMM3. Although LMM2 did not present the greatest Marginal R 2 value, the difference from the others is minimal. All the models had similar conditional values of Conditional R², suggesting that the overall explanatory power of the models remains unaffected by the addition of new predictor variables. For this reason, LMM2 provides a good balance between model fit and the inclusion of relevant predictors ( Table 3 and S1 ) . View this table: View inline View popup Download powerpoint Table S1: Comparison of the various linear mixed-effect models performed View this table: View inline View popup Download powerpoint Table 3: The effect of commensal bacteria to pneumococcal growth of 28 strains of S. pneumoniae using linear mixed-effect model. Pneumococcal strain variability The results of the LMM2 revealed considerable variability in pneumococcal growth among strains. The intraclass correlation coefficient (ICC) was 0.55, indicating that 55% of the total variability in growth could be attributable to differences between individual strains. The variance of the random effects between strains was 0.178, while the variance between replicates within the same strain was lower, 0.063 ( Table 3 ). These findings showed that a considerable portion of the observed variability in pneumococcal growth was accounted for by differences between individual strains rather than variation among replicates of the same strain. Inhibitory effect of Dolosigranulum pigrum and Corynebacterium pseudodiphtheriticum on pneumococcal growth The results of LMM2 analysis provided strong evidence of the association between the culture condition and the pneumococcal growth, although pneumococcal strain variability was observed. The presence of D. pigrum was significantly associated with a reduction in pneumococcal growth, as reflected by the estimate coefficient (β = −0.763, CI: −0.94 to −0.59, p < 0.0001). Similarity C. pseudodiphtheriticum exhibited a notable inhibitory effect, (β = −0.583, CI: −0.76 to −0.41, p < 0.0001). Notably, the combined presence of both bacteria was strongly associated (β = −0.971, CI: −1.15 to −0.79, p < 0.0001), with pneumococcal growth decrease. Even though D. pigrum and C. pseudodiphtheriticum showed an individual inhibitory effect on pneumococcal growth, their combined effect was even significantly stronger, with estimated coefficients of −0.207 (CI: −0.39 to −0.03, p < 0.01) and −0.387 (CI: −0.57 to −0.21, p < 0.0001) when compared to co-culture with D. pigrum alone or C. pseudodiphtheriticum alone, respectively ( Figure 2 and Table 3 ) . Download figure Open in new tab Figure 2: Pneumococcal growth under different culture conditions: Cultured alone (red, labeled as S for Streptococcus ), cultured with D. pigrum (orange, labeled as SD), cultured with C. pseudodiphtheriticum (light green, labeled as SC), and cultured with both commensal bacteria (dark blue, labeled as SDC). The data is represented by boxplots showing the median and interquartile range (IQR) of pneumococcal growth (measured in gc/¿l after log-transformation), along with individual data points (n=338). Black points denote outliers. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 . Variation in pneumococcal growth according to strain features In the LMM analysis, the site of isolation did not indicate any significant association with pneumococcal growth (β = −0.079, CI = −0.48 to 0.33, p = 0.708) ( Figure 3C and Table S1 ) . Furthermore, pneumococcal growth appeared not to be generally affected by the invasiveness of pneumococcal serotype. low invasive disease potential serotypes pneumococcal isolates showed a slightly reduced growth, although not statistically significant, overall compared to high invasive disease potential serotypes (β = −0.250, CI = −0.60 to −0.10, p = 0.175) ( Figure 3A and Table 3 ) . Moreover, a detailed subanalysis examining this predictor under different culture conditions revealed that low invasive disease potential serotypes isolates showed a greater decrease in growth when they were cultured with both commensal bacteria (β = −0.355, CI = −0.76 to 0.05, p = 0.082) than when they were cultured alone, where the effect was minimal (β = −0.112, CI = −0.52 to 0.29, p = 0.577). However, this effect was not significant, indicating no clear association between these variables. ( Figure 3B and Table S2 ) . View this table: View inline View popup Download powerpoint Table S2: Linear mixed-effect model to analyse the effect of Jnvasiveness of serotype in presence or absence of commensal bacteria to pneumococcal growth Download figure Open in new tab Figure 3: Pneumococcal growth according to strain features. A) Invasiveness of pneumococcal serotypes, HIPST (light blue) vs LIPST (dark blue). B) Invasiveness of pneumococcal serotypes in different culture conditions. C) Site of isolation, NPA (purple) vs blood (yellow). The data is represented by boxplots showing the median and interquartile range (IQR) of pneumococcal growth (measured in gc/¿L after log-transformation), along with individual data points (n=338). Black points denote outliers. *p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 DISCUSSION The present study evaluated the effect of two commensal bacteria from the nasopharyngeal microbiota on the growth of different pneumococcal strains as well as other characteristics of the strain itself, such as serotype invasiveness and the isolation site of the isolate, which may influence this effect. The main findings of this study indicated that despite the variability among pneumococcal strains, the presence of those two commensal bacteria, alone or in combination, had a substantial impact on pneumococcal growth. Specifically, the growth of S. pneumoniae was significantly decreased in the presence of D. pigrum, supporting its potential interest as a candidate for inclusion in a probiotic formula. An ideal probiotic for the respiratory microbiota should be a non-pathogenic bacterium that is present as a natural carrier, capable of adhering to the epithelium and colonising the niche of key pathobionts. It also should have no cytotoxic effect on respiratory epithelial cells, resistance against the horizontal gene transfer and mobile genetic elements, a low tendency for tissue invasion, and be susceptible to commonly used antibiotics ( 19 , 26 ). Recent reviews of the literature highlight these characteristics of D. pigrum , supporting its potential as a probiotic candidate ( 19 ). Nonetheless, even with these promising features, the current published studies have been confined to very few strains and have not considered the extensive genetic diversity of S. pneumoniae, which consists of 100 serotypes and thousands of clones with different invasive disease potential ( 1 , 2 , 4 , 45 ). One of the key strengths of this study is the use of various strains of S. pneumoniae with different intrinsic characteristics, such as distinct serotypes, and isolated from different anatomical sites. This diversity in pneumococcal strains allowed considering strain-specific differences in the pneumococcal growth in the analysis, providing stronger insights into potential interactions. The analysis conducted on S. pneumoniae strain-specific variables revealed that the site of isolation (nasopharynx or blood) and serotype invasiveness did not significantly affect the antagonistic effect of commensal bacteria in pneumococcal growth. These findings suggest that the inhibition of S. pneumoniae is consistent regardless of its site of isolation and invasiveness, enhancing the capacity of D. pigrum as a potential probiotic. However, the limited number of strains emphasises the importance of additional studies to validate this consistent antagonistic effect and thus better comprehend the underlying interactions. The exact mechanisms by which D. pigrum exerts its observed antagonistic effect on pneumococcal growth remain unclear. One possible explanation is competition of nutrients, which are frequently a limiting factor for bacteria colonisation, such as S. pneumoniae. Therefore, S. pneumoniae experiences reduced nutritional availability when it coexists with other bacteria, which consequently restricts its growth ( 46 ). Another factor could be the secretion of antimicrobial compounds, such as bacteriocins, which can function as a bactericidal or bacteriostatic agent against pathogens ( 23 , 24 ). Specifically, it is suggested that D. pigrum may produce lantipeptides, a type of bacteriocin with notable antimicrobial activity that can disrupt bacteria’s cell walls, restricting pathogen proliferation ( 47 – 49 ).Thus, pneumococcal inhibition may be explained in part by D. pigrum’s antimicrobial characteristics. In order to comprehend the precise association between D. pigrum and S. pneumoniae and the role of bacteriocins in this instance, additional analyses are crucial. D. pigrum as a LAB, has the ability to generate lactate in the nasal microbiome. Lactate is an acid that can lower the environmental pH, creating unfavourable conditions for the optimal growth of S. pneumoniae , which grows in a more neutral pH ( 50 ). However, prior studies such as those by Brugger S, et al., indicate that D. pigrum ’s lactate production for itself is insufficient to fully inhibit pneumococcal growth ( 14 ). This suggests that additional inhibition mechanisms are taking place. An interesting aspect of the results is the synergistic effect observed when another commensal bacterium was present. Specifically, the inhibition of S. pneumoniae growth was significantly greater when D. pigrum cooperated with C. pseudodiphtheriticum . Although both bacteria individually already showed inhibitory effects, their combined effect is significantly stronger. This suggests that the interaction between D. pigrum and C. pseudodiphtheriticum enhances the reduction of pneumococcal growth. This observation could be indicative of a synergistic interaction between these two commensal bacteria, which aligns with the study of Brugger S. In this regard, D. pigrum is auxotrophic for certain nutrients, especially for aminoacids, and it is hypothesised that C. pseudodiphtheriticum may provide these nutrients, enhancing their mutual inhibitory effects on S. pneumoniae ( 14 , 51 , 52 ). A synergistic effect of bacteriocins from different bacterial isolates cannot be ruled out either. Some limitations should be noted in the study. First, only a single strain of D. pigrum and C. pseudodiphtheriticum was used for the in vitro study, which limits the generalisability of the results. It is possible that other strains of these commensal bacteria may not have the same inhibitory effect on S. pneumoniae . Also, the results obtained should be considered in the context of previous findings on the dynamics of bacterial inhibition, where the order of exposure between commensal and pathogenic bacteria plays a crucial role ( 53 ). In this study, a bacteria invasion scenario, where a pathogen attempts to colonise a pre-existing respiratory microbiota, was simulated by adding S. pneumoniae to a previously grown culture of both commensal bacteria. Future studies could explore more scenarios to determine to what extent the order affects the ability of commensal bacteria to inhibit respiratory pathogens. Furthermore, a few of the observed associations did not reach statistical significance, which could be explained by the limited sample size. Future research should consider increasing the sample size and incorporating genomic and phenotypic studies to improve the accuracy and elucidate the possible mechanism of action. These findings could have important implications for understanding microbial interactions within polymicrobial environments, particularly in the context of bacterial interference and competition within the respiratory tract. The observed inhibitory effects suggest that these commensal bacteria may play a protective role in mitigating pneumococcal replication. The antagonistic effect of these commensal bacteria on S. pneumoniae replication supports the potential protective factor of a healthy nasopharyngeal microbiota against IPD and underscores the potential of these microorganisms as promising probiotic candidates. ACKNOWLEDGES The authors acknowledge the financial assistance extended by the projects FIS PI19/00104 and PI23/00049, and to PFIS fellowship FI 24/00206. This support was crucial for the attainment of results presented in the present research. AUTHOR CONTRIBUTIONS Conceptualization: C.MA and M.BF; Data curation: M.C; Formal analysis: M.C and M.BF; Investigation: M.C; Methodology: C.MA, P.B, D.H, A.L and A.M; Resources: D.H, A.PA and P.C; Supervision: C.MA and M.BF; Writing-original draft: C.MA, M.C and M.BF; Writing-review and editing: A.L, P.B, D.H, A.PA, P.C, G. GC and A.M. REFERENCES 1. ↵ Bogaert D , De Groot R , Hermans PWM. 2004 . Streptococcus pneumoniae colonisation: the key to pneumococcal disease . Lancet Infect Dis 4 : 144 – 154 . doi: 10.1016/S1473-3099(04)00938-7 OpenUrl CrossRef PubMed Web of Science 2. ↵ Wahl B , O’Brien KL , Greenbaum A , Majumder A , Liu L , Chu Y , Lukšić I , Nair H , McAllister DA , Campbell H , Rudan I , Black R , Knoll MD . 2018 . 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Share Inhibitory effect of Dolosigranulum pigrum and Corynebacterium pseudodiphtheriticum on pneumococcal in vitro growth M Cisneros , M Blanco-Fuertes , A Lluansí , P Brotons , D Henares , A Pérez-Argüello , G González-Comino , P Ciruela , A Mira , C Muñoz-Almagro bioRxiv 2025.01.10.632320; doi: https://doi.org/10.1101/2025.01.10.632320 Share This Article: Copy Citation Tools Inhibitory effect of Dolosigranulum pigrum and Corynebacterium pseudodiphtheriticum on pneumococcal in vitro growth M Cisneros , M Blanco-Fuertes , A Lluansí , P Brotons , D Henares , A Pérez-Argüello , G González-Comino , P Ciruela , A Mira , C Muñoz-Almagro bioRxiv 2025.01.10.632320; doi: https://doi.org/10.1101/2025.01.10.632320 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7622) Biochemistry (17650) Bioengineering (13871) Bioinformatics (41881) Biophysics (21424) Cancer Biology (18566) Cell Biology (25461) Clinical Trials (138) Developmental Biology (13365) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15590) Genomics (22476) Immunology (17713) Microbiology (40331) Molecular Biology (17148) Neuroscience (88473) Paleontology (666) Pathology (2827) Pharmacology and Toxicology (4816) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)