Features of the upper respiratory tract microbiota in bacterial carriage of Streptococcus pneumoniae in patients with metabolic syndrome

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Introduction

The human upper respiratory tract (URT) is a complex ecosystem that exists in dynamic equilibrium with the microbiota [1, 2]. The homeostasis of this system is maintained by a variety of factors, including mucociliary clearance, secretion of antimicrobial peptides (e.g., defensins, lysozyme), immune surveillance (IgA, macrophages, neutrophils), and competitive interactions between microorganisms [3, 4]. Disruption of this balance caused by external (infections, antibiotics) or internal (metabolic, immune disorders) factors can lead to dysbiosis, increasing the risk of respiratory and systemic diseases [5, 6].

Normally, the microbiota of the URT is represented by commensal bacteria that suppress the colonization of pathogens by competing for resources and producing bacteriocins [7, 8]. However, when conditions change (e.g., decreased local immunity, inflammation), opportunistic microorganisms (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus) capable of causing infections begin to predominate [9, 10].

S. pneumoniae colonizes the nasopharynx of 20–60% of healthy adults [11], while remaining the leading pathogens of invasive infections, including pneumonia, meningitis, and acute otitis media [12–14]. The level and duration of its carriage are determined by the complex interaction of three key factors. First, competitive interaction with other members of the microbiota is critical. For example, S. aureus is capable of suppressing pneumococcal colonization [15], while H. influenzae competes for the same epithelial cell receptors [16]. Second, the immune status of the macroorganism has a significant impact. It has been established that secretory IgA deficiency is associated with an increased frequency and density of S. pneumoniae colonization [17]. Finally, systemic factors, including metabolic disorders, play an important role. A number of studies demonstrate a link between obesity, diabetes mellitus, dyslipidemia, and changes in the nature of pneumococcal carriage [18, 19]. This complex nature of interactions makes the study of S. pneumoniae carriage particularly relevant for understanding the pathogenesis of respiratory infections and developing preventive strategies.

Despite significant progress in studying the relationship between gut microbiota and metabolic syndrome (MetS) [20], the impact of this pathological condition on respiratory tract microbial communities remains insufficiently studied. Currently, there are no comprehensive studies in the scientific literature that directly demonstrate the relationship between MetS and the characteristics of S. pneumoniae colonization, as well as the accompanying changes in the composition of the respiratory microbiota.

Of particular scientific interest is the question of S. pneumoniae carriage in patients with MetS [21, 22], as well as the composition of the URT microbial community [6, 10]. Furthermore, the task of identifying reliable biomarkers of dysbiotic changes in the microbiota of the URT in patients with metabolic disorders remains relevant [23, 24].

The aim - to study the effect of metabolic syndrome in carriers of Streptococcus pneumoniae on the composition of the upper respiratory tract microbiota community

Materials and methods

A prospective case-control study was conducted. It included 171 S. pneumoniae carriers, who were divided into two groups: group 1 — 118 patients with MetS; group 2 — 53 patients without MetS. The study was conducted with the voluntary informed written consent of the patients. The study protocol was approved by the Ethics Committee of Samara State Medical University (Protocol No. 254 dated September 28, 2022).

The diagnosis of MetS was established according to the modified criteria of the International Diabetes Federation, which included the presence of abdominal obesity (waist circumference ≥ 94 cm for men and ≥ 80 cm for women) in combination with two or more additional criteria: hypertension (blood pressure ≥ 130/85 mmHg or antihypertensive therapy), fasting hyperglycemia (glucose ≥ 5.6 mmol/L) or insulin resistance (HOMA-IR ≥ 2.5), as well as dyslipidemia (triglycerides ≥ 1.7 mmol/L or high-density lipoproteins < 1.0 mmol/L for men/< 1.3 mmol/L for women).

Inclusion criteria:

• age ≥ 21 to 55 years;
• confirmed carriage of pneumoniae;
• absence of antibiotic therapy within 90 days prior to sample collection;
• no history of vaccination against pneumococcal infection;
• no chronic lung disease (chronic obstructive pulmonary disease, bronchial asthma), immunosuppression (HIV) or acute infections when included into the study;
• no exacerbation of chronic infections;
• no active herpes infection;
• no history of glucocorticosteroid use.

Clinical material was collected using sterile swabs with Amies transport medium. A swab was taken from the oropharynx and nasopharynx of each participant. The swabs were immediately delivered to the laboratory within 2 hours for further processing. The samples were seeded on 5% blood agar and selective blood agar with gentamicin added. The plates were incubated in 5% CO₂ at 37°C for 24–48 hours. Presumptive identification of colonies morphologically similar to S. pneumoniae was based on characteristic alpha-hemolysis, sensitivity to optochin, and the presence of a lysis reaction with 10% bile.

Species identification of all isolates was confirmed by mass spectrometry (MALDI-TOF MS). The results were interpreted in accordance with the manufacturer's recommendations (logarithmic score ≥ 2.0 for reliable species-level identification). The number of CFU/swab was determined by serial dilutions.

The degree of colonization was classified as:

• low: 10¹–10² CFU/swab;
• medium: 10³–10⁴ CFU/swab;
• high: 10⁵–10⁶ CFU/swab.

In the second stage, high-throughput sequencing of the 16S rRNA gene was used for taxonomic analysis of the microbiota of the upper respiratory tract. Total genomic DNA was extracted from clinical samples using commercial reagent kits. Amplification of the hypervariable regions V3–V4 of the 16S rRNA gene was performed using universal primers, followed by library preparation and sequencing on the Illumina platform in accordance with the manufacturer's standard protocols.

Bioinformatics data processing was performed using standard bioinformatics protocols. After quality control, clustering into operational taxonomic units was performed with a similarity threshold of 97%. Taxonomic identification was performed using 16S rRNA reference databases.

Quantitative indicators between groups were compared using parametric and nonparametric criteria, and qualitative variables were analyzed using the χ² criterion. Statistical significance was determined at a level of p < 0.05.

Results

Analysis of the quantitative level of S. pneumoniae colonization revealed differences between the groups examined. In group 1, low colonization (10¹–10² CFU/swab) was detected in 24 (20.3%) individuals, moderate colonization (10³–10⁴ CFU/swab) in 43 (35.6%), and high colonization (10⁵–10⁶ CFU/swab) in 51 (43.2%) individuals; in the second group, these figures were 27 (51%), 18 (34%), and 8 (15%), respectively. A comparative analysis showed that the proportion of individuals with a high degree of bacterial colonization in the MetS group was statistically significantly higher than in the control group (χ² = 16.6; p < 0.001).

Nasopharyngeal microbiota

Analysis of the composition of the nasopharyngeal microbiota in individuals carrying S. pneumoniae revealed differences between groups with and without MetS. In both groups, the nasopharyngeal microbiota was predominantly composed of Firmicutes, Actinobacteriota, and Proteobacteria, but their relative proportions differed significantly. In patients in group 2, Firmicutes (35%) and Actinobacteriota (28%) dominated, while in patients in group 1, there was a shift toward an increase in the proportion of Proteobacteria (36% vs. 22%; p < 0.01) with a simultaneous decrease in Actinobacteriota (16% vs. 28%; p < 0.01). The proportion of Firmicutes in MetS was slightly lower (30% vs. 35%), but the difference was not statistically significant.

Analysis at the genus level showed that in individuals in group 2, the leading taxa were Streptococcus (20%), Corynebacterium (18%), and Dolosigranulum (10%), which are associated with colonization resistance and a stable microbial ecosystem. In group 1, there was a significant decrease in the content of Corynebacterium (9% vs. 18%; p < 0.01) and Dolosigranulum (4% vs. 10%; p < 0.01) against the background of an increase in Moraxella (18% vs. 12%; p = 0.02), Haemophilus (12% vs. 8%; p = 0.04), and Staphylococcus (8% vs. 4%; p = 0.05).

Analysis of 16S rRNA sequencing followed by taxonomic assignment allowed us to characterize the microbial community of the nasopharynx. In a number of taxa, species identification was possible with a high degree of reliability. Both commensal and opportunistic microorganisms were identified in the analyzed samples of both groups. Among the dominant taxa identified to the species level in individuals of the second group were S. mitis (18.6%), Corynebacterium accolens (18.4%), D. pigrum (14.1%), and M. catarrhalis (12.6%). Less represented were H. parainfluenzae (10.3%), S. aureus (9.2%), and Neisseria subflava (9.9%). It should be noted that the accuracy of species identification based on the 16S rRNA gene varies for a number of closely related bacteria (e.g., representatives of the S. mitis/pneumoniae/oralis complex); this approach does not always allow for unambiguous species differentiation. The microbial profile of group 1 was characterized by a significant increase in the proportion of opportunistic pathogens for which species identification was achieved. Among them, the most common were S. mitis (21.9%; p = 0.041), M. catarrhalis (17.5%; p = 0.033), H. parainfluenzae (15.2%; p = 0.027), and S. aureus (14.8%; p = 0.039). The proportion of C. accolens (11.7%; p = 0.045) and D. pigrum (9.8%; p = 0.038) was lower than in individuals without MetS, while N. subflava accounted for 9.1% (p = 0.062) (Fig. 1).

 

Fig. 1. Taxonomic composition of the nasopharynx microbiota at the species level in patients in groups 1 (outer circle) and 2 (inner circle), %.

 

Oropharyngeal microbiota

Analysis of the structure of the microbial community of the oropharynx at the species level showed that Firmicutes, Bacteroidetes and Proteobacteria dominated in both groups. In group 2, the proportions of these taxa were 52.4, 25.7 and 18.3%, respectively, and in group 1, they were 48.1, 20.2 and 27.9%. Thus, participants with MetS showed a relative increase in Proteobacteria and a decrease in Bacteroidetes, which is consistent with the literature data on dysbiotic changes in metabolic disorders.

At the genus level, Streptococcus (38.5%), Prevotella (19.6%), and Veillonella (14.8%) predominated in individuals without MetS, forming a structure characteristic of conditionally healthy oropharyngeal microbiota. In individuals with MetS, there was a shift towards an increase in the proportion of Haemophilus (17.4%) and Neisseria (15.9%) with a relative decrease in Prevotella to 12.1%, while Streptococcus remained dominant (35.1%).

Analysis of 16S rRNA allowed for detailed characterization of the oropharyngeal microbiota, revealing the presence of both commensal and opportunistic microorganisms, some of which were reliably identified to species level. In patients with MetS, the dominant taxa identified at the species level were predominantly N. subflava (18.2% vs. 14.7% in individuals without MetS; p = 0.04) and H. parainfluenzae (15.5% vs. 13.2%; p = 0.05), which may indicate a tendency toward an increase in the proportion of Gram-negative microorganisms. Opportunistic pathogens such as Fusobacterium nucleatum (8.4% vs. 6.8%; p = 0.03) were also more frequently identified in the MetS group (Fig. 2). It should be noted that the accuracy of species identification based on the 16S rRNA gene varies: for a number of closely related bacteria (e.g., representatives of the Streptococcus genus or various species of Neisseria), this approach does not always allow for unambiguous species differentiation.

 

Fig. 2. Taxonomic composition of the oropharynx microbiota at the species level in patients in groups 1 (outer circle) and 2 (inner circle), %.

Fig. 2. Taxonomic composition of the oropharynx microbiota at the species level in patients in groups 1 (outer circle) and 2 (inner circle), %.

 

At the same time, individuals without MetS had a slightly higher average relative abundance of Gemella haemolysans (14.5% vs. 12.1%; p = 0.12), Granulicatella adiacens (10.3% vs. 9.1%; p = 0.20), and Leptotrichia buccalis (7.6% vs. 6.1%; p = 0.07), although these differences did not reach statistical significance.

Discussion

Our study shows that S. pneumoniae carriers with a higher bacterial load were significantly more common in the group of patients with MetS. It is known that high colonization density increases the risk of pneumococcal transmission and the development of invasive forms of infection [25]. The results obtained suggest that metabolic disorders accompanied by chronic inflammation and reduced effectiveness of local immune barriers create conditions for more intense pneumococcal persistence [26].

At the same time, patients with MetS were more likely to have colonization not only with S. pneumoniae, but also with other opportunistic microorganisms. This is probably due to the fact that systemic metabolic disorders, such as obesity, insulin resistance, and dyslipidemia, indirectly affect the microbial community of the upper respiratory tract by weakening the barrier function of the mucous membranes and dysregulating the immune response [19, 27, 28].

In the nasopharynx of patients with MetS, an increase in the proportion of H. parainfluenzae, M. catarrhalis and S. aureus was observed, with a simultaneous decrease in the content of C. accolens and D. pigrum. It is known that C. accolens and D. pigrum are involved in the formation of colonization resistance by inhibiting the growth of pathogens, including pneumococcus [29]. A decrease in the proportion of these protective taxa may reduce the resistance of the microbial community to the introduction of potential pathogens.

N. subflava, H. parainfluenzae and F. nucleatum predominated in the oropharynx of individuals with MetS. A number of studies indicate that F. nucleatum is capable of forming biofilms in the oral cavity and oropharynx [30], contributing to the persistence of pathogens and the maintenance of chronic inflammation.

MetS is characterized by chronic inflammation, hormonal changes (including increased leptin and decreased adiponectin levels), oxidative stress and impaired lipid and carbohydrate metabolism [28]. These factors can alter the expression of mucins and antimicrobial peptides [4] and affect the adhesion and growth of microorganisms on the mucous membranes of the respiratory tract.

Similar changes have been described in studies of the nasopharyngeal microbiota in obese adults, where a decrease in Corynebacterium and Dolosigranulum and an increase in Haemophilus and Moraxella were also observed [23].

The data obtained emphasize the need for further research to assess the impact of MetS on the composition of the upper respiratory tract microbiota.

Conclusion

1. Among S. pneumoniae carriers with MetS, the proportion of individuals with a high degree of bacterial colonization of the nasopharynx is higher (43.2% versus 15.0% in the control group).
2. The nasopharyngeal microbiota of S. pneumoniae carriers with MetS is characterized by a decrease in the proportion of commensal taxa (Corynebacterium, Dolosigranulum) and an increase in the proportion of opportunistic microorganisms (M. catarrhalis, H. parainfluenzae, S. aureus).
3. Analysis of the oropharyngeal microbiota of S. pneumoniae carriers showed that individuals with MetS exhibit signs of dysbiotic changes, manifested by an increase in the proportion of Proteobacteria due to N. subflava and H. parainfluenzae, as well as more frequent detection of opportunistic pathogens, including F. nucleatum. In individuals without MetS, the microbial community was characterized by a large representation of commensal taxa (Gemella haemolysans, Granulicatella adiacens, Leptotrichia buccalis).

About the authors

Valeria A. Starikova

Samara State Medical University

Author for correspondence.
Email: v.a.starikova@samsmu.ru
ORCID iD: 0009-0006-5483-1784

Assistant Professor, Department of infectious diseases with epidemiology

Russian Federation, Samara

Dmitry Yu. Konstantinov

Samara State Medical University

Email: d.u.konstantinov@samsmu.ru
ORCID iD: 0000-0002-6177-8487

Dr. Sci. (Med.), Head, Department of infectious diseases with epidemiology

Russian Federation, Samara

Elena A. Konstantinova

Samara State Medical University

Email: e.a.konstantinova@samsmu.ru
ORCID iD: 0000-0002-6022-0983

Cand. Sci. (Med.), Associate Professor, Department of infectious diseases with epidemiology

Russian Federation, Samara

References

Supplementary files