Graphical abstract

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Highlights

     * •
       SNP patterns shift from healthy to disease stages, impacting
       pathogenicity
     * •
       bGWAS shows 32% of genes change from colonization to pneumonia
     * •
       Peptidoglycan pathway links to pneumonia pathogenicity
     * •
       PBP3 mutations for β-lactam resistance differ in health and
       pneumonia
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   Disease; Genomics; Immunity

Introduction

   Childhood severe pneumonia is a serious respiratory disease with a high
   incidence and mortality rate among children in developing countries. To
   date, pneumonia remains one of the leading causes of death among all
   children who die before the age of five years as per etiologic
   investigations.[38]^1^,[39]^2 The most common pathogens causing
   childhood pneumonia include Streptococcus pneumoniae, Haemophilus
   influenzae type b (Hib), respiratory syncytial virus (RSV), and
   Mycoplasma pneumoniae.[40]^3^,[41]^4 In the past, invasive diseases
   caused by Haemophilus influenzae were often linked to strains with type
   b polysaccharide capsules. However, since the introduction of the Hib
   conjugate vaccine, there has been a significant decrease in the
   frequency of illnesses caused by this particular
   serotype.[42]^5^,[43]^6 Worldwide, there has been a significant
   increase in invasive infections caused by non-vaccine-preventable
   Haemophilus influenzae strains. This increase is mainly attributed to
   NTHi strains.[44]^7^,[45]^8

   NTHi is an opportunistic pathogen of the upper respiratory tract in
   healthy children, which can infect the lower respiratory tract and
   induce chronic pulmonary diseases. Studies have shown that NTHi is the
   most commonly isolated bacterial pathogen in otitis media and sinusitis
   in children and one of the major drivers of acute exacerbation of
   chronic obstructive pulmonary disease (COPD) in adults.[46]^9^,[47]^10
   NTHi colonization/infection is also common in young children with
   cystic fibrosis.[48]^11

   The molecular mechanisms behind the transition of NTHi from a commensal
   organism to a pathogen remain unclear. This shift may involve both
   internal changes within the organism and adaptations within the host.
   At the host level, when immune function is compromised, normally benign
   colonizers like NTHi can proliferate excessively and release
   significant amounts of inflammatory mediators, leading to infectious
   diseases. In this context, the bacteria are referred to as
   "opportunistic pathogens." Reduced immune function is thus a critical
   factor in the transition of NTHi from a commensal to a pathogenic
   state. For example, one study observed that even mild influenzae A
   virus infections can impair the host’s specific TH17 response to NTHi,
   increasing susceptibility to secondary bacterial infections.[49]^12

   Further research has focused on bacterial strain variations. Some
   studies suggest that phase variation in NTHi is a mechanism underlying
   changes in its virulence. This includes homologous recombination,
   variations in simple sequence repeat lengths between allele variants,
   phase variation mediated by slip-strand mispairing, and transcriptional
   termination due to frameshift mutations.[50]^13^,[51]^14^,[52]^15 Some
   studies have shown that the phase variation of specific
   lipooligosaccharide (LOS) biosynthesis genes plays a critical role in
   the transition from colonizing the human nasopharynx to invading the
   middle ear cavity during the course of otitis media.[53]^16 Overall,
   there are relatively few genomic studies on the differences between
   NTHi from a commensal organism to a pathogen, partly because it is
   difficult to obtain pathogenic samples.

   The present study aims to explore the genomic differences among NTHi
   strains isolated from the nasopharynx swabs of healthy children and
   bronchoalveolar lavage fluids (BALFs) of children with acute or chronic
   pneumonia. The objective is to uncover the genomic characteristics that
   enable NTHi to adapt to pulmonary infections and the underlying
   mechanisms through which NTHi causes acute and chronic pneumonia in
   children. This investigation is critical for enhancing the diagnosis,
   treatment, and prevention of NTHi-related lower respiratory tract
   diseases.

Results

Heterogeneity of NTHi genome across different clinical phenotypes

   The experimental design is outlined in [54]Figure 1A. This study
   sequenced 69 samples to investigate the genomic characteristics of NTHi
   across different clinical phenotypes. The samples comprised 23 NTHi
   isolates from children with acute pneumonia, 27 from children with
   chronic pneumonia, and 19 from healthy children for comparison.
   Pneumonia samples (50 total) were obtained from BALF, whereas control
   samples (19 total) were collected from nasopharyngeal swabs.
   Next-generation sequencing yielded high-quality data with an average
   coverage depth of 1,161x ([55]Table S1).

Figure 1.

   [56]Figure 1
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   Sample collection workflow for phylogenetic tree analysis

   (A) Sample collection flowchart; 69 NTHi samples were acquired from
   this study, and 52 Haemophilus influenzae (Hi) reference sequences were
   sourced from the NCBI database.

   (B) A combination NJ tree of samples and HI references, present as Fan