# Exploring Competitive Relationship Between Haemophilus parainfluenzae and Mitis Streptococci via Co-Culture-Based Molecular Diagnosis and Metabolomic Assay

## TL;DR

This study explores how Haemophilus parainfluenzae and mitis Streptococci compete in saliva, using co-culture experiments and metabolomic analysis to understand their interactions and impact on oral health.

## Contribution

The study provides novel insights into the competitive relationship between H. parainfluenzae and mitis Streptococci through co-culture assays and metabolomic profiling.

## Key findings

- H. parainfluenzae maintains nitrate reduction ability in co-culture but with reduced efficiency compared to monoculture.
- qRT-PCR confirms competitive growth between H. parainfluenzae and S. australis and S. sanguinis.
- Metabolomic analysis suggests metabolic compounds from mitis Streptococci drive inter-strain competition.

## Abstract

Various bacterial strains with nitrate-reducing capacity (NRC), such as Haemophilus, Actinomyces, and Neisseria, are known to promote NH3 production, control pH in the oral cavity, and inhibit the growth of aciduric bacteria. However, experimental evidence on various estimated bacterial networks within the salivary microbiome is insufficient. This study aims to explore potential bacterial compositional competition observed within saliva samples from dental caries patients through a co-culture assay of mitis Streptococci, which is a primary colonizer in the salivary microbiome, and nitrate-reducing bacteria Haemophilus parainfluenzae. We investigated bacterial growth efficiency change by co-culture time using the qRT-PCR method. In addition, we applied LC/Q-TOF-based metabolites screening to confirm metabolic interactions between oral bacterial species and their association with dental caries from a metabolomics perspective. As a result, we first found that the nitrate reduction ability of H. parainfluenzae is maintained even in a co-culture environment with the mitis Streptococci group through a nitrate reduction test. However, nitrate reduction efficiency was hindered when compared with monoculture-based nitrate reduction test results. Next, we designed species-specific primers, and we confirmed by qRT-PCR that there is an obvious competitive relationship in growth efficiency between H. parainfluenzae and two mitis Streptococci (S. australis and S. sanguinis). Furthermore, although direct effects of nitrate reduction on competition have not been identified, we have potentially confirmed through LC/Q-TOF-based metabolite screening analysis that the interaction of various metabolic compounds synthesized from mitis Streptococci is driving inter-strain competition. In particular, we constructed a basic reference core-metabolites list to understand the metabolic network between each target bacterial species (H. parainfluenzae and mitis Streptococci) within the salivary microbiome, which still lacks accumulated research data. Ultimately, we suggest that our data have potential value to be referenced in further metagenomics and metabolomics-based studies related to oral health care.