https://orcid.org/0009-0001-9715-887X
Figures
Abstract
Background
Respiratory tract infections range from asymptomatic colonisation to an invasive disease. Recent studies suggest that nasopharyngeal microbiota may influence this variability. Emerging evidence points to Dolosigranulum pigrum, a nasopharyngeal commensal, as a potentially protective bacterium. This study aimed to identify variables associated with the presence of D. pigrum in the nasopharynx of children with varying respiratory health statuses.
Methods
Nasopharyngeal aspirates were collected from children <18 years who were asymptomatic (n = 65), had banal viral infection (n = 48), or Invasive Pneumococcal Disease (IPD) (n = 27). The presence of D. pigrum was defined as >0.1% of total sequences obtained by 16S rRNA gene sequencing. Variables included sex, breastfeeding, delivery mode, S. pneumoniae carriage, respiratory viruses and clinical features.
Results
Among 140 children (73 males, 67 females), D. pigrum was detected in 79 (56.4%): 44/65 in the healthy group; 26/48 of viral and 9/27 IPD cases. Multivariate analysis revealed significant associations with health status and sex. Healthy children were more likely to carry D. pigrum than IPD cases (44/79 vs. 26/79; p = 0.028). Males were more frequently D. pigrum carriers than females (48/79 vs. 31/79; p = 0.033).
Citation: Cisneros M, Henares D, Lluansí A, Brotons P, Launes C, Penela-Sanchez D, et al. (2026) Presence of Dolosigranulum pigrum in the nasopharynx and its relationship with respiratory health status in paediatric population. PLoS One 21(9): e0345189. https://doi.org/10.1371/journal.pone.0345189
Editor: Shahina Akter, Bangladesh Council of Scientific and Industrial Research, BANGLADESH
Received: March 9, 2026; Accepted: July 8, 2026; Published: September 2, 2026
Copyright: © 2026 Cisneros et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The data underlying this study are available in public repositories. Raw sequence files have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB41741. Associated datasets, including metadata, OTU sequences, OTU tables, taxonomic classifications, phylogenetic tree, and supplementary materials, are available in the Figshare repository at https://doi.org/10.6084/m9.figshare.13280435.v3 Additional data are provided within the Supporting Information files of the original study and this paper.
Funding: This work was supported by the Instituto de Salud Carlos III (PI13/01729, PI16/00174, PI19/00104, and PI23/00049 to CMA), and by the PFIS fellowship (FI24/00206 awarded to MC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Carmen Muñoz-Almagro reports travel grants from Pfizer, MSD, and personal fees as speaker from Sanofi-Pasteur and MSD. The rest of authors declare no conflicts of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Introduction
Respiratory tract infections (RTIs) remain a major public health challenge, with lower respiratory tract infections (LRTIs) being the leading cause of death worldwide, especially in children under five years [1]. These infections are commonly caused by pathobionts such as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, or by respiratory viruses. Pathobionts possess a dual potential role: they can asymptomatically colonise the nasopharynx, but under certain conditions, they may transition to causing local diseases or invade normally sterile anatomical sites, leading to invasive disease [2]. S. pneumoniae is one major respiratory pathogen due to the high burden of invasive pneumococcal disease (IPD) [2,3].
Emerging evidence suggests that the human respiratory microbiota composition could be an important factor influencing the different behaviours of nasopharyngeal pathobionts [4]. The respiratory microbiota comprises diverse microbial communities that colonise the respiratory tract. These communities appear to play an important role in protecting against or increasing the risk of some infectious diseases. Nasopharyngeal microbiome dysbiosis, characterised by a decrease in microbial diversity and depletion of commensal bacteria that normally prevent pathogenic overgrowth, can lead to increased susceptibility to infection [5–7]. Specifically, certain studies have identified the Corynebacterium and Dolosigranulum species as significant beneficial members of the nasopharynx microbiota [8–14].
Dolosigranulum pigrum first described by Aguirre, et al. in 1993 [15] is a commensal bacterium from the upper respiratory tract commonly isolated from the nasal cavity and nasopharynx [15]. This Gram-positive coccus is a lactic acid bacterium that is normally susceptible to beta-lactams [16]. Extensive evidence supports D. pigrum as a beneficial respiratory commensal. Microbiota studies have reported that respiratory health is strongly associated with the detection of the genus Dolosigranulum in the nasopharynx and the nasal cavity [17,18]. Conversely, absence or low abundance of D. pigrum has been linked to a wide variety of respiratory infections, including otitis, sinusitis, bronchitis, bronchiolitis, and pneumonia, as well as exacerbations of chronic respiratory diseases. In particular, our group has demonstrated this association extended beyond respiratory infections to invasive disease caused by S. pneumoniae [19,20].
Despite epidemiological evidence linking D. pigrum to respiratory health, the mechanisms remain poorly understood. It has been postulated an antagonism with the main respiratory pathogens. In vitro studies have demonstrated inhibition of pneumococcal growth by D. pigrum, though specific mechanisms have not been fully elucidated [14,21]. Additionally, D. pigrum appears to modulate respiratory innate immunity and enhance resistance to pathogens such as Respiratory Syncytial Virus (RSV) and S. pneumoniae [22–25]. Notably, nasal administration of D. pigrum has been shown to decrease the number of S. pneumoniae and reduce its spread to the blood [23].
In light of the substantial evidence supporting the potential benefits and protective role of D. Pigrum in relation to respiratory health, it is essential to understand the factors that influence its presence in the nasopharynx. Therefore, the aim of the present study was to identify and analyse the epidemiological, clinical and microbiological variables that may be associated with the presence or absence of D. pigrum in the nasopharynx of children under the age of 18 years with different respiratory health statuses.
Materials and methods
Study design
This ancillary study used data from a previously case-control study at Sant Joan de Déu Barcelona Children’s Hospital (HSJD) from January 2014 to December 2018. The cohort included three paediatric groups according to health status: (i) healthy/asymptomatic outpatients; (ii) outpatients with microbiologically verified banal symptomatic viral RTIs; and (iii) inpatients with IPD. No statistically significant differences were observed among groups regarding age, sex, or seasonality [20].
The present analysis did not involve new participant recruitment or additional sample collection. Data for the current secondary analysis were accessed in 2025. The dataset was de-identified prior to analysis, and the authors did not have access to information that could identify individual participants.
The inclusion criteria were: (i) meeting the case/control definition previously detailed [20] (ii) having informed consent signed by the parents or legal guardians of participants; (iii) participants not belonging to a previously defined clinical risk group for developing IPD [26]; and (iv) no antibiotic exposure before sample collection (or ≤24 h for IPD cases) [27,28].
Data collection
Diverse epidemiological, clinical, and microbiological variables were collected from all participants. Epidemiological variables included age, sex, ethnicity, delivery mode, maternal breastfeeding, pneumococcal vaccination status, and others. Clinical parameters were only applicable to IPD cases and included length of hospital stay, admission and length to the Paediatric Intensive Care Unit (PICU), and clinical manifestations. Microbiological parameters included pneumococcal colonisation status, pneumococcal nasopharyngeal load, invasive disease potential of nasopharyngeal pneumococcal serotypes, composition of the nasopharyngeal bacterial microbiota, and respiratory viral detection. All the variables collected were previously described [20].
Sample collection
Nasopharyngeal aspirates (NPAs) were collected from all the participants included in the study. The procedure to collect these samples was previously published [20]. The samples were kept at −80°C until the laboratory analyses were conducted.
Pneumococcal detection, quantification, and serotyping
A duplex real-time PCR targeting the lytA gene of S. pneumoniae and RNase P, a human control gene that identifies sample viability and inhibitors, was used in NPAs samples for detection of S. pneumoniae. Primers and probes were utilised according to the Centers for Disease Control and Prevention (CDC) guidelines [29].
Respiratory virus detection
The multiplex real-time PCR Allplex II RV16 detection kit (Seegene) was used to detect the DNA/RNA of 15 of the most frequent human respiratory viruses on NPAs [30].
Variable definition
In the current study, the main outcome was the presence or absence of D. pigrum in NPA, which was determined by bacterial 16S rRNA gene sequencing in the preceding main study [20]. The sequences assigned to Dolosigranulum spp. were analysed and compared to the total sequences to calculate the relative abundance of D. pigrum. D. pigrum was considered to be present in NPAs if it comprised >0.1% of the total sequences. The presence of D. pigrum was correlated with epidemiological, clinical, and microbiological variables previously collected. The serotypes 1, 3, 4, 5, 7F, 8, 9A, 9V, 12F, 14, 18C, 19A, and 33F were considered as high-invasiveness disease potential serotypes, as previously reported [20].
For subanalyses of relative abundance of D. pigrum, the healthy group was further stratified based on respiratory virus detection status into: (i) healthy virus-negative (asymptomatic without respiratory virus detection) and (ii) healthy virus-positive carriers (asymptomatic with respiratory virus detection).
Statistical analyses
Statistical analyses were performed using the 4.4.2 version of R and RStudio software [31] using car [32], MASS [33], emmeans [34] and lmtest [35] packages. Shapiro-Wilk test was used to evaluate the normality of the data. Significance of associations between the presence or absence of D. pigrum with categorical variables was tested by Chi-Square or Fisher’s test. Fisher’s test was used according to Cochran rule, if ≥20% of cells had expected frequencies <5. Associations of D. pigrum presence or absence with continuous variables were analysed using the Student t-test or the Mann-Whitney test for normally or not normally distributed data, respectively.
In order to assess potential relationships between the epidemiological, clinical, and microbiological variables and the presence of D. pigrum, a generalised linear model (GLM) with a binomial distribution and a logistic link function was used. An initial model was built up, including all the variables that showed an association with the presence of D. pigrum at a p-value ≤0.2 and did not have collinearity. A stepwise approach combining forward and backward selection was used to gradually add and eliminate variables to refine the optimal model. This strategy was based on the Akaike Information Criterion (AIC), which balances model fit and complexity [36]. To determine collinearity among the variables, the Variance Inflation Factor (VIF) was calculated, and the variables with a VIF > 5 were considered collinear and excluded from the final model [37].
As a complementary quantitative analysis to assess the effect of abundance beyond presence/absence (qualitative analysis), the relative abundance of D. pigrum across respiratory health status groups was evaluated by the Kruskal-Wallis test, followed by the pairwise Wilcoxon test. This analysis included all the participants. Relative abundance values were log10-transformed prior to analysis.
Associations were measured by adjusted Odds Ratios (aORs) with corresponding 95% confidence intervals (CI). Estimated Marginal Means assessed the significance of the associations. A p-value <0.05 was established to determine statistical significance.
Ethical approval statement
This is an ancillary study of data from a previously published study [20].The original research was approved by the Ethics Committee of Hospital Sant Joan de Déu (PIC 70−15 and PIC 137−16) and conducted in compliance with the Helsinki Declaration and Spanish regulations on data protection (Organic Laws 15/1999 and 14/2007). Written informed consent was obtained from all parents/legal guardians in the original study. Data used in the present analysis were anonymized.
Results
Characteristics of study population
A total of 140 participants were selected for the study, of which D. pigrum was detected in 79 (56.4%) (median age: 33.6 months [IQR: 2.0–41.5]) and absent in 61 (median age: 38.5 months [IQR: 0.1–48.0]). Detection rates were similar between children under two years of age (36/62, 58.1%) and older children (43/78, 55.1%). Regarding respiratory health status, D. pigrum was found in 44/65 (67.7%) in healthy children, 26/48 (54.2%) in viral cases and 9/27 (33.3%) in IPD cases (Fig 1A, Table 1). The proportion of D. pigrum carriers differed significantly across healthy and IPD groups (p = 0.002).
This is the Fig 1 legend: (A) Proportion of D. pigrum carriers in healthy children, viral and IPD cases. Numbers above bars indicate carrier/total of each group. Differences assessed by Chi-square test. (B) Log-transformed relative abundance of D. pigrum in healthy children, viral and IPD cases. (C) Extended comparison including healthy subgroup. Box plots shown median, interquartile range and individual values. Statistical comparisons performed using Kruskal-Wallis test followed by pairwise Wilcoxon tests. P-values are indicated above brackets.
Among D. pigrum carriers, males predominated (48/79, 60.8%) over females (31/79, 39.2%). Most children were born via vaginal delivery (D. pigrum carriers: 69.7%, non-carriers: 68.4%) and were breastfed for more than six months (D. pigrum carriers: 69.7%; non-carriers: 68.2%). In relation to the vaccination status, a higher proportion of children with D. pigrum were vaccinated (74.7%) than unvaccinated (63.9%). Kindergarten attendance was slightly below 50% in both groups (Table 1).
Regarding clinical features, healthy children represented a proportion of 55.7% in the group with D. pigrum and 34.4% in the group without D. pigrum. In contrast, IPD represented a proportion of 29.5% among children without D. pigrum (Table 1). Among IPD cases, all the children with D. pigrum and the majority of the children without D. pigrum presented pneumonia (100.0% and 77.8%, respectively) (Table 2).
From a microbiological perspective, among participants with the presence of D. pigrum, 62.8% were carriers of S. pneumoniae, with LRST accounting for 83.7% in the group with D. pigrum. Additionally, the children without D. pigrum had a 70.5% of pneumococcal carriage (Table 3). Respiratory viruses were detected in 70.7% of D. pigrum carriers and 86.9% in non-carriers. Rhinovirus/Enterovirus was the most frequent virus found in both groups, followed by Bocavirus (21.4%) in the D. pigrum-positive group and Adenovirus (28.3%) in the D. pigrum-negative group (Table 3).
Association of the presence of D. pigrum with epidemiological, clinical and microbiological variables
Significant differences were observed between males and females in the presence of D. pigrum, with males being more likely than to have D. pigrum in the nasopharynx (OR: 2.2, 95% CI: 1.12–4.43; p = 0.02). The association remained significant in the multivariate analysis, with an adjusted odds ratio (aOR) of 2.21 (95% CI: 1.07–4.6; p = 0.03). The multivariate generalized lineal model included 4 variables (respiratory health status, sex, maternal breastfeeding and respiratory virus detection), selected through an AIC-based stepwise procedure. Children who were not breastfed had a lower probability of having D. pigrum compared to those who were breastfed, but the difference between these proportions was not statistically significant (univariate, p = 0.15 and multivariate p = 0.06). No significant associations were found between D. pigrum colonisation and age group, type of delivery, vaccination status and kindergarten attendance.
The univariate analysis revealed a significant association of the respiratory health status and D. pigrum colonisation, with healthy children more likely to carry D. pigrum compared with those with IPD (OR, 4.1; 95% CI: 1.6–11.5; p = 0.003). This association remained significant in the multivariate analysis, with an aOR of 3.7 (95% CI: 1.1–12.6; p = 0.028). Both univariate and multivariate analyses showed no significant differences were observed between healthy children and viral cases (univariate, p = 0.15 and multivariate p = 0.86) or between viral and IPD cases (univariate analysis: p = 0.09; multivariate analysis: p = 0.10) (Table 4).
Absence of respiratory viruses was associated with a higher proportion of D. pigrum’s presence in the univariate analysis (OR: 2.7; 95% CI: 1.1–6.9; p = 0.02), but not in the multivariate analysis (p = 0.06). The distribution of individual respiratory viruses did not differ significantly across D. pigrum carriers and non-carriers. No significant differences were found in relation to the presence of D. pigrum according to S. pneumoniae carrier status (Table 4).
Differential abundance of D. pigrum across respiratory health status groups
Relative abundance of D. pigrum was evaluated across all participants as a complementary analysis to assess the effect of the abundance beyond presence/absence. This analysis showed significantly higher abundance of D. pigrum among healthy children compared to IPD cases (p = 0.001) (Fig 1B). Viral cases exhibited an intermediate level of D. pigrum abundance, with significant differences compared to healthy children (p = 0.029) but not when compared to IPD cases (p = 0.06). Further subclassification of healthy children revealed significant differences in D. pigrum abundance between subgroups (p = 0.003), with the healthy virus-negative group showing the highest abundance (Fig 1C). This group also showed significantly higher D. pigrum abundance than viral cases (p = 0.001) and IPD cases (p = 0.0002). In contrast, no significant differences were observed between healthy virus-positive carriers and either viral or IPD cases.
Discussion
This study examined epidemiological, clinical, and microbiological variables associated with Dolosigranulum pigrum presence in the paediatric nasopharynx during the pre-pandemic period. Given the emerging evidence of the potential protective role in respiratory health of D. pigrum [8–14], the study included different study groups according to different respiratory health statuses to provide a comprehensive analysis.
Results demonstrated a significant association between D. pigrum presence and male sex, with males more likely to have nasopharyngeal D. pigrum colonisation. This finding sheds more evidence of sex-based differences in bacterial composition [38,39]. Previous research has suggested that the human microbiome is influenced by hormonal, genetic, and behavioural factors [40,41], which vary between sexes. These differences could impact the composition of the respiratory microbiota, potentially allowing D. pigrum to play a compensatory role in protecting against pathogens in males, who are known to have a higher risk of lower respiratory tract infections (LTRIs) [40,42]. Although this hypothesis has not been deeply investigated, it raises the possibility that the presence of D. pigrum in males could represent an evolutionary ecological adaptation to balance sex disparities in disease susceptibility. Furthermore, the lower prevalence of S. pneumoniae carriers among males reported in a retrospective cohort study [43] could contribute to the higher prevalence of D. pigrum in this study group, given their antagonistic relationship [14,21]. Despite these insights, research on sex-related microbiota differences remains in its early stages, with limited evidence currently available. The present study contributes to this emerging area of research by providing additional evidence of sex-related microbiome variations.
Moreover, the findings revealed a significant association with the presence of D. pigrum and respiratory health status. Specifically, healthy children were more likely to have D. pigrum compared to IPD cases. These findings are aligned with the hypothesis that D. pigrum may play a protective role against IPD, a condition caused by the invasion of S. pneumoniae into sterile territories [2,3]. As previously reported pneumococcal colonisation is considered a major risk for IPD [2], consequently in this study S. pneumoniae carrier status was assessed in all participants. Although a lower proportion of pneumococcal carriers was noted among children with D. pigrum, the association did not reach statistical significance. This suggests that the protective role of D. pigrum may extend beyond inhibiting pneumococcal colonisation, as previously reported [14,21]. It is plausible that D. pigrum employs additional mechanisms that impede the transition of S. pneumoniae from a colonising to an invasive state, therefore playing a role in preventing IPD. These mechanisms could include interactions with other microbiome components [14,21], modulation of the host immune response [22] or the production of metabolites that interfere with the virulence of S. pneumoniae [44–46]. Furthermore, the analysis of D. pigrum relative abundance provided additional evidence to these findings supporting this protective role. Healthy children not only had higher prevalence of D. pigrum but also showed greater abundance compared to IPD cases. This dose-dependent pattern suggests that the colonisation density of D. pigrum might be related with its protective role, with higher bacterial loads potentially conferring stronger resistance against progression to invasive disease.
In contrast, regarding respiratory health status, no significant association was observed between D. pigrum presence and viral infection. In addition, the association between the presence of D. pigrum and PCR-confirmed detection of respiratory viruses was evaluated. This analysis demonstrated that children with D. pigrum were less likely to have respiratory viruses. Although this association reached statistical significance in the univariate analysis, it was not found in the multivariate analysis, remaining as a non-significant trend. This attenuation effect in the multivariate model may reflect the influence of confounding variables, suggesting that the univariate association could be explained by other covariates rather than a direct role of D. pigrum presence. However, across all study groups, respiratory virus infections were banal or asymptomatic. This result aligns with previous reports suggesting that severe respiratory infections are typically associated with the absence of D. pigrum [25]. However, the relative abundance analysis showed that the presence of respiratory virus significantly affects D. pigrum colonisation, resulting in lower abundance. This finding suggests that respiratory viruses influence the colonisation density of D. pigrum in the nasopharynx. Nevertheless, all these findings highlight the compelling necessity of future studies to clarify the potential role of D. pigrum in modulating the respiratory microbiome and its implications for paediatric respiratory health.
The present study has some limitations. Firstly, the sample size of the IPD case study group was smaller compared to the other study groups. This limitation derives from the selection criterion of including only cases with less than 24 hours of antibiotic exposure. Many potential IPD cases were excluded because they had received antibiotic treatment for longer periods before sample collection, which would have compromised the reliability of the results. Given that D. pigrum is a Gram-positive coccus highly sensitive to antibiotics such as beta-lactams [16], prolonged antibiotic exposure could have reduced its detectable presence, leading to false-negative results. Therefore, ensuring this criterion was crucial to maintain the reliability and robustness of the findings. Furthermore, the small number of positive D. pigrum events in the IPD subgroup resulted in a low Events Per Variable ratio. Although the stepwise AIC-based variable selection was employed to minimise overfitting by reducing model complexity, the limited sample size of the IPD subgroup may affect the precision of the estimates and the generalisability of the findings for this group. Nevertheless, the associations identified in the multivariate model were consistent with the univariate analyses and with previously reported findings, supporting the biological plausibility of the results despite this constraint. However, the reduced sample size of our study highlights the need for future studies with larger cohorts to confirm and expand these observations. Secondly, the relative abundance of D. pigrum was calculated as the proportion of reads specifically assigned to this specific taxon relative to the total number of reads obtained by sequencing the V3-V4 region of the 16S rRNA gene. While this approach is common in microbiota studies, it presents specific challenges, such as the low taxonomic resolution of the 16S rRNA gene amplicon, which may limit the accurate identification of D. pigrum at the species level. Therefore, future studies could complement this approach with 3rd generation full-length 16S sequencing [47], WGS metagenomics data or quantitative techniques (such as qPCR) to validate and refine the determination of bacterial presence. Thirdly, although previous studies have demonstrated a synergistic effect between D. pigrum and C. pseudodiphtheriticum in the reduction of pneumococcal growth [14], the present study focused exclusively on D. pigrum. Future studies should explore these bacteria interactions to better understand the complex ecological dynamics that may contribute to respiratory health.
In summary, the present study provides further evidence supporting the protective role of D. pigrum against IPD, highlighting its potential as a key modulator of paediatric respiratory health. These findings reinforce the importance of future studies to elucidate the underlying mechanisms and to evaluate the potential for leveraging D. pigrum in preventive or therapeutic strategies against severe respiratory infections.
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