Background
Wheezing is a common respiratory symptom in children, especially in infants and young children, and about one-third of children will have at least one episode of wheezing by the age of 3 years, and 1–2% of children are hospitalized for severe respiratory symptoms [
1]. Wheezing resolves with age in some infants and children, while others develop persistent wheezing or asthma [
2]. Recurrent wheezing in infants and children is often attributed to airway injury, inflammation, and immune responses due to infection, allergy, and environmental factors. However, few reports have shown whether bacterial flora disorders in the lower respiratory tract are associated with recurrent wheezing in infants and children. The human lower respiratory tract’s bacterial load is at least several times lower than that of the other parts of the body and may be highly plastic and susceptible to external forces [
3,
4].
The earliest description of symbiotic microorganisms in the body can be traced back to the study of
Enterobacter in the digestive tract of infants by Escherich more than a century ago [
5]. Over the decades, the microbiome’s importance has gradually gained recognition, from early descriptions of gut bacteria in response to diet to the detailed mechanisms of host-microbe interactions found in a large body of work documented in recent years, including pathways affecting systemic immunity, cardiovascular health, and neurological development [
6‐
9]. However, research and understanding of the bacterial community structure of the lung remain scarce as it was long considered a sterile environment, except when infected by respiratory pathogenic microbes. This deeply-rooted notion led to the removal of lungs from the sampling list in the first round of the Human Microbiome Project (HMP) [
10]. The second round of the HMP found viruses and fungi in the lung in addition to bacteria, but the health and disease effects of each microorganism are still not well understood [
11‐
14].
In recent years, recurrent wheezing in infants and preschoolers has become very common. The increase in emergency department visits and hospitalization has societal as well as personal effects [
15]. The prognosis of recurrent wheezing in infants and children varies, and the onset and duration of wheezing may sometimes be inevitably associated with asthma [
16,
17], which has been proven to be caused by impaired lung function due to bacterial infection.
Streptococcus pneumoniae,
Moraxella catarrhalis, and
Haemophilus influenzae are the microbiota primarily associated with the exacerbation of asthma [
18,
19]. However, it remains unclear whether prolonged, persistent wheezing versus multiple wheezing episodes cause different changes in the lower respiratory tract’s colony structure.
The ideal method of specimen collection is to collect lung tissue samples by isolating microbial deoxyribonucleic acid (DNA) from host DNA, but this method cannot be routinely applied to every disease. Although sputum specimens are easy to collect, contact with the oral cavity and upper respiratory tract is unavoidable during the collection process. In contrast, bronchoalveolar lavage fluid is immune to upper airway flora contamination and closely mimics the lower airway’s microbiome but cannot wholly sample the lung’s deepest airways. Bronchial foreign bodies are a typical pediatric emergency, commonly affecting infants and children aged six months to three years. Most children with bronchial foreign bodies have a short duration of illness, and mechanical damage to the airway should not adjust the microbiota’s composition over this short period. Therefore, we chose the results of alveolar lavage fluid from children with bronchial foreign bodies instead of lower airway bacterial colonization from healthy infants and children as a control.
Because persistent and repeated wheezing may cause changes in the colony structure of lower respiratory tract microorganisms, the differences in the genera represented by the lower respiratory tract microbiota in infants and children with recurrent wheezing should be elucidated to provide a basis for future insights into the critical role of lower respiratory tract bacteria in the development of recurrent wheezing in infants and children.
Therefore, the first aim of this study was to describe the composition and diversity of bacterial colonies in the lower respiratory tract of children with recurrent wheezing. The second aim was to determine whether persistent wheezing affects infants' and children's microbial colony structure and diversity. The third objective was to identify the dominant bacterial genera in infants and children with recurrent wheezing, provide new ideas for future insights into the role of bacterial genera in regulating airway immunity, and provide new directions for the prevention of diseases that cause recurrent wheezing.
Discussion
Humans have evolved symbiotically with microorganisms, and host-microbial interactions are gradually being elucidated, with bacterial colonization of the human host impacting a variety of diseases [
28]. Microbial colonization can be detected in the naso- and oropharynx of newborns within the first 5 min after birth, indicating that colonization of the upper respiratory tract has begun [
29]. Initially, the immune system of newborns will adjust so that the microbiota can colonize the respiratory tract smoothly. The stable symbiotic microbiota will gradually promote the improvement of the neonatal immune system, and the establishment of this microbiota gives the body its ability to fight pathogenic bacteria. The resident microbiota and the infant’s immune system are restricted in many ways and regulated by metabolites and microorganisms. Immunoglobulin A, present in breast milk, can help establish a stable host-microbiota relationship [
30]. Bacteria, viruses, and fungi are the primary pathogens responsible for human respiratory infections. However, in 30% of respiratory cases, pathogens remain undetected by conventional methods [
31].
Data released in 2017 suggest that the bacterial microbiota differs slightly between bronchial lavage fluid and endotracheal samples [
32]. Our analysis of alveolar lavage fluid microbiomes from 48 children showed that the lower respiratory flora of those in the wheezing group (showing both recurrent and persistent wheezing) comprised mainly the phylum Proteobacteria, followed by the thick-walled Firmicutes and the Bacteroidota.
In contrast, the alveolar lavage fluid specimens from children in the foreign body group (control group) were mainly composed of the phylum Firmicutes, followed by Proteobacteria and Bacteroidota. There is a relative lack of data on children’s lower respiratory flora, and studies of oropharyngeal microflora have confirmed that the phyla mentioned above were found to be dominant in both asthmatic and healthy populations [
33,
34]. A neonatal-based study showed that
H. influenzae,
M. catarrhalis, and
S. pneumoniae were associated with wheezing outcomes within the first 5 years of life. Although there is no direct evidence for the cellular impact of these bacterial fixations, these associations may be consistent with significant neutrophil elevation in children with severe recurrent wheezing [
18,
35].
We compared the wheezing and foreign body groups in terms of microbial genera and observed abundant Streptococcus spp. in each group. The numbers of bacteria of genera Sphingomonas and Phyllobacterium were significantly higher in the recurrent wheezing group than in the control group; Phyllobacterium spp. were also higher in the prolonged wheezing group than in the control group, and Prevotella spp., Neisseria spp., and Haemophilus spp. were higher in the control group than in the wheezing groups.
The low proportions of
Moraxella spp.,
Staphylococcus spp., and
Haemophilus spp. in the wheezing group were different from the increased proportions of
Staphylococcus spp.,
Moraxella spp., and
Haemophilus spp., which have been previously reported to increase the risk of chronic wheezing in children [
35‐
40], and require further investigation. The high proportion of
Phyllobacterium spp. has not been reported to be associated with wheezing in infants and children. However, Wen et al. found a higher enrichment of
Phyllobacterium foliaris among oropharyngeal microorganisms in children with the influenza A virus; the exact mechanism remains unclear [
41].
Reduced numbers of
Prevotella spp. were found in both wheezing groups.
Prevotella spp. is a common commensal fixer at mucosal sites and is the dominant respiratory genus [
42]. The homeostatic role of
Prevotella spp. in the healthy lung remains largely unclear. Inflammation and pathology in the chronic obstructive pulmonary disease-like lungs of mice induced by lipopolysaccharide or lipopolysaccharide elastase inhalation were reduced by this genus and mediated the growth of
Pseudomonas and
Lactobacillus spp. This suggests that the reduction of
Prevotella spp. (possibly by creating a microenvironment unsuitable for the growth of the genus, which leads to a decrease in its abundance) in patients with wheezing may not be a pre-disease risk factor.
Some authors have compared the inflammatory properties of
Prevotella spp. in the lungs of healthy individuals with those of bacilli associated with pulmonary disease in asthmatic patients (
H. influenzae B,
H. influenzae, and
M. catarrhalis).
Prevotella was found to induce similar levels of CD83, CD86, and CD40 activation marker surface expression, but compared with Proteobacteria, it reduced the levels of interleukin (IL)-12p70, IL-23, and IL-10 cytokines in monocyte-derived dendritic cells. The lower inflammatory capacity of
Prevotella was also confirmed in mice, where
Prevotella reduced the production of macrophage inflammatory protein 2-α (IL-8), tumor necrosis factor-a (TNF-a), and thymic stromal lymphopoietin by lung stromal cells, as well as the production of TNF-a by lung immune cells, suggesting that
Prevotella may be well tolerated in the lungs [
43‐
45].
A high proportion of
Streptococcus spp. was found in all three groups, and it has been shown that increased abundance of
Moraxella and
Streptococcus spp. in nasopharyngeal swabs from infants with capillary bronchitis is associated with recurrent wheezing in children at 3 years of age [
46]. This may suggest that infant wheezing is more likely to result from a disruption of overall bacterial microecology and host respiratory and immune homeostasis rather than the influence of a single bacterium. Bacterial detection in the lower airway may be due to an imbalance between constant exposure to upper respiratory microorganisms through micro-inhalation and clearance by endothelial cilia and coughing in the respiratory tract.
The exact reasons for the differences in the dominant genera are unclear and may lead to molecular changes in the mucin glycoproteins during long-term chronic inflammation; mucin glycans are a significant source of nutrients and adhesion sites for bacteria and fungi in diseased airways, and different microorganisms can utilize specific mucins, which influence airway fixation [
47]. The physical and chemical factors in the airway (including, oxygen tension, pH, temperature, and mucus) vary with the airway’s anatomical position [
48], which, together with irregular medication use in children with multiple and persistent wheezing, may lead to the establishment of new, stable, and complex microflora in the presence of immune dysregulation.
The microbial diversity of the wheezing group was lower than that of the foreign body group, and the diversity of microbial colonies was reduced. The exact mechanism by which bacteria cause wheezing in infants and children remains unclear. The immune balance between the bacteria and host organism may be disrupted. Bacteria and their products (such as endotoxins) can stimulate the production of cytokines in airway epithelial cells. Some cytokines (such as nuclear factor kappa B) can activate the IL-8 gene and then initiate positive feedback regulation to increase the production of neutrophils and inflammatory factors, such as IL-8, IL-6, and TNF-α. The increase in TNF-α production can increase the reactivity of the airway’s smooth muscles, thereby aggravating airway obstruction and causing wheezing [
49‐
51].
Pulmonary function tests can help in judging the severity of diseases with wheezing as a symptom and also in evaluating clinical efficacy and prognosis [
52]. The most commonly used lung function test for infants and young children is the tidal breathing lung function test [
53]. Both TPEF/TE and VPEF/VE are important parameters reflecting obstructive ventilation disorders, with a normal range of 28–55%. Depending on the degree of obstruction, it is classified as mild obstruction (23–28%), moderate obstruction (15–22%), and severe obstruction (< 15%) [
54]. The pulmonary function of the children we observed with wheezing suggested a decrease in severe obstructive ventilation. However, the mean TPEF/TE and VPEF/VE in the multiple wheezing groups were worse than those in the persistent wheezing group, and we speculated that perhaps the persistent wheezing was due to persistent airway spasm caused by some infection, while the more severe pulmonary impairment in the recurrent wheezing group may have caused a greater chance of asthma. However, why the duration of disease differs in patients with similar colony structure is subject to further research. We speculate that this may be related to insufficient and ineffective treatment of infants or children.
The main advantage of our study is that the matched case–control excludes the deviation caused by the influence of age, time, and sex. We also elucidated that the duration of wheezing can affect the structure of the bacterial microbial community. We used alveolar lavage fluid samples obtained via bronchoscopy, which is closer to the proper lower respiratory tract microbial colony composition, and the bronchoscopic foreign body group as the control, which is closer to the community composition of respiratory bacteria in children of the same age.
However, our study had some limitations. We primarily focused on the bacterial group of microorganisms and could not exclude the possibility that viruses and fungi might be involved in recurrent wheezing in children. As in other observational studies, our findings do not necessarily suggest causality. 16S rRNA sequencing allows the annotation between genus- and species-level identification of bacteria but does not provide the resolution of macrogenomic techniques (e.g., birdshot sequencing), especially for closely related species, such as Streptococcus spp. and Prevotella spp. Specific species may need further discussion.
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