{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T22:58:47Z","timestamp":1787785127321,"version":"build-2784847793"},"reference-count":35,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2025,1,26]],"date-time":"2025-01-26T00:00:00Z","timestamp":1737849600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Education of the Republic of Korea","award":["RS-2024-00355393"],"award-info":[{"award-number":["RS-2024-00355393"]}]},{"name":"National Research Foundation of Korea","award":["RS-2024-00355393"],"award-info":[{"award-number":["RS-2024-00355393"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/microorganisms13020279","type":"journal-article","created":{"date-parts":[[2025,1,27]],"date-time":"2025-01-27T06:39:51Z","timestamp":1737959991000},"page":"279","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Exploring Competitive Relationship Between Haemophilus parainfluenzae and Mitis Streptococci via Co-Culture-Based Molecular Diagnosis and Metabolomic Assay"],"prefix":"10.3390","volume":"13","author":[{"given":"Yeseul","family":"Choi","sequence":"first","affiliation":[{"name":"Department of Microbiology, College of Bio-Convergence, Dankook University, Cheonan 31116, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinuk","family":"Jeong","sequence":"additional","affiliation":[{"name":"Smart Animal Bio Institute, Dankook University, Cheonan 31116, Republic of Korea"},{"name":"Center for Bio-Medical Core Facility, Dankook University, Cheonan 31116, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Youngjong","family":"Han","sequence":"additional","affiliation":[{"name":"Department of Microbiology, College of Bio-Convergence, Dankook University, Cheonan 31116, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miyang","family":"Han","sequence":"additional","affiliation":[{"name":"Department of Microbiology, College of Bio-Convergence, Dankook University, Cheonan 31116, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Byungsun","family":"Yu","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, College of Bio-Convergence, Dankook University, Cheonan 31116, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6791-2408","authenticated-orcid":false,"given":"Kyudong","family":"Han","sequence":"additional","affiliation":[{"name":"Department of Microbiology, College of Bio-Convergence, Dankook University, Cheonan 31116, Republic of Korea"},{"name":"Smart Animal Bio Institute, Dankook University, Cheonan 31116, Republic of Korea"},{"name":"Center for Bio-Medical Core Facility, Dankook University, Cheonan 31116, Republic of Korea"},{"name":"Department of Human Microbiome Research HuNbiome Co., Ltd., R&D Center, Seoul 08507, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Cugini, C., Ramasubbu, N., Tsiagbe, V.K., and Fine, D.H. (2021). Dysbiosis From a Microbial and Host Perspective Relative to Oral Health and Disease. Front. Microbiol., 12.","DOI":"10.3389\/fmicb.2021.617485"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Wei, S., Bahl, M.I., Baunwall, S.M.D., Hvas, C.L., and Licht, T.R. (2021). Determining Gut Microbial Dysbiosis: A Review of Applied Indexes for Assessment of Intestinal Microbiota Imbalances. Appl. Environ. Microbiol., 87.","DOI":"10.1128\/AEM.00395-21"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1038\/s41368-022-00163-7","article-title":"Oral microbiota in human systematic diseases","volume":"14","author":"Peng","year":"2022","journal-title":"Int. J. Oral Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1111\/prd.12393","article-title":"The oral microbiome: Role of key organisms and complex networks in oral health and disease","volume":"87","author":"Sedghi","year":"2021","journal-title":"Periodontology 2000"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Zheng, H., Xie, T., Li, S., Qiao, X., Lu, Y., and Feng, Y. (2021). Analysis of oral microbial dysbiosis associated with early childhood caries. BMC Oral Health, 21.","DOI":"10.1186\/s12903-021-01543-x"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Liu, S., Xie, G., Chen, M., He, Y., Yu, W., Chen, X., Mao, W., Liu, N., Zhang, Y., and Chang, Q. (2023). Oral microbial dysbiosis in patients with periodontitis and chronic obstructive pulmonary disease. Front. Cell. Infect. Microbiol., 13.","DOI":"10.3389\/fcimb.2023.1121399"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Maier, T. (2023). Oral Microbiome in Health and Disease: Maintaining a Healthy, Balanced Ecosystem and Reversing Dysbiosis. Microorganisms, 11.","DOI":"10.3390\/microorganisms11061453"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Jeong, J., Ahn, K., Mun, S., Yun, K., Kim, Y.-T., Jung, W., Lee, K.E., Kim, M.-Y., Ahn, Y., and Han, K. (2023). Understanding the bacterial compositional network associations between oral and gut microbiome within healthy Koreans. J. Oral Microbiol., 15.","DOI":"10.1080\/20002297.2023.2186591"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1007\/s13258-024-01526-6","article-title":"Exploring oral bacterial compositional network in two oral disease groups using a convergent approach of NGS-molecular diagnostics","volume":"46","author":"Jeong","year":"2024","journal-title":"Genes Genom."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Sato-Suzuki, Y., Washio, J., Wicaksono, D.P., Sato, T., Fukumoto, S., and Takahashi, N. (2020). Nitrite-producing oral microbiome in adults and children. Sci. Rep., 10.","DOI":"10.1038\/s41598-020-73479-1"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/j.niox.2019.10.012","article-title":"Effects of age, sex and diet on salivary nitrate and nitrite in infants","volume":"94","author":"Timby","year":"2020","journal-title":"Nitric Oxide"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Rosier, B.T., Moya-Gonzalvez, E.M., Corell-Escuin, P., and Mira, A. (2020). Isolation and Characterization of Nitrate-Reducing Bacteria as Potential Probiotics for Oral and Systemic Health. Front. Microbiol., 11.","DOI":"10.3389\/fmicb.2020.555465"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1159\/000529162","article-title":"The Role of Oral Nitrate-Reducing Bacteria in the Prevention of Caries: A Review Related to Caries and Nitrate Metabolism","volume":"57","author":"Feng","year":"2023","journal-title":"Caries Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/B978-0-12-800261-2.00002-5","article-title":"Community interactions of oral streptococci","volume":"87","author":"Jakubovics","year":"2014","journal-title":"Adv. Appl. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Dashper, S.G., Mitchell, H.L., Le Cao, K.-A., Carpenter, L., Gussy, M.G., Calache, H., Gladman, S.L., Bulach, D.M., Hoffmann, B., and Catmull, D.V. (2019). Temporal development of the oral microbiome and prediction of early childhood caries. Sci. Rep., 9.","DOI":"10.1038\/s41598-019-56233-0"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1111\/1348-0421.13028","article-title":"Oral mitis group streptococci: A silent majority in our oral cavity","volume":"66","author":"Okahashi","year":"2022","journal-title":"Microbiol. Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"L\u2019Heureux, J.E., van der Giezen, M., Winyard, P.G., Jones, A.M., and Vanhatalo, A. (2023). Localisation of nitrate-reducing and highly abundant microbial communities in the oral cavity. PLoS ONE, 18.","DOI":"10.1371\/journal.pone.0295058"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1016\/j.ijmm.2013.08.006","article-title":"Haemophilus parainfluenzae expresses diverse lipopolysaccharide O-antigens using ABC transporter and Wzy polymerase-dependent mechanisms","volume":"303","author":"Young","year":"2013","journal-title":"Int. J. Med. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"6249","DOI":"10.1128\/AEM.00347-09","article-title":"Inhibition of nitrate reduction by chromium (VI) in anaerobic soil microcosms","volume":"75","author":"Kourtev","year":"2009","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"102790","DOI":"10.1016\/j.anaerobe.2023.102790","article-title":"Growth assessment of mixed cultures of probiotics and common pathogens","volume":"84","author":"Stapleton","year":"2023","journal-title":"Anaerobe"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1016\/j.jfca.2018.08.002","article-title":"Peptide biomarkers for identifying the species origin of gelatin using coupled UPLC-MS\/MS","volume":"73","author":"Ward","year":"2018","journal-title":"J. Food Compos. Anal."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1031","DOI":"10.1099\/00221287-136-6-1031","article-title":"Naturally occurring NAD-independent Haemophilus parainfluenzae","volume":"136","author":"Gromkova","year":"1990","journal-title":"J. Gen. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1016\/j.jobcr.2012.07.001","article-title":"Oral microbial habitat a dynamic entity","volume":"2","author":"Tanwir","year":"2012","journal-title":"J. Oral Biol. Craniofacial Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1","DOI":"10.2334\/josnusd.45.1","article-title":"Evaluation and comparison of lactobacilli characteristics in the mouths of patients with or without cavities","volume":"45","author":"Ahumada","year":"2003","journal-title":"J. Oral Sci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"177","DOI":"10.4161\/21505594.2014.970472","article-title":"The novel species Streptococcus tigurinus and its association with oral infection","volume":"6","author":"Zbinden","year":"2015","journal-title":"Virulence"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Yoon, B.K., Jackman, J.A., Valle-Gonzalez, E.R., and Cho, N.-J. (2018). Antibacterial Free Fatty Acids and Monoglycerides: Biological Activities, Experimental Testing, and Therapeutic Applications. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19041114"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Baishya, J., Bisht, K., Rimbey, J.N., Yihunie, K.D., Islam, S., Al Mahmud, H., Waller, J.E., and Wakeman, C.A. (2021). The Impact of Intraspecies and Interspecies Bacterial Interactions on Disease Outcome. Pathogens, 10.","DOI":"10.3390\/pathogens10020096"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1128\/MMBR.67.3.429-453.2003","article-title":"Surviving the acid test: Responses of gram-positive bacteria to low pH","volume":"67","author":"Cotter","year":"2003","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/j.jgar.2020.01.022","article-title":"Minocycline, focus on mechanisms of resistance, antibacterial activity, and clinical effectiveness: Back to the future","volume":"22","author":"Asadi","year":"2020","journal-title":"J. Glob. Antimicrob. Resist."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1021","DOI":"10.1007\/s11745-016-4174-5","article-title":"Traumatic Acid Reduces Oxidative Stress and Enhances Collagen Biosynthesis in Cultured Human Skin Fibroblasts","volume":"51","author":"Pankiewicz","year":"2016","journal-title":"Lipids"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"558","DOI":"10.1016\/j.ijantimicag.2014.01.031","article-title":"Species-level assessment of the molecular basis of fluoroquinolone resistance among viridans group streptococci causing bacteraemia in cancer patients","volume":"43","author":"Sahasrabhojane","year":"2014","journal-title":"Int. J. Antimicrob. Agents"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Okahashi, N., Nakata, M., Sumitomo, T., Terao, Y., and Kawabata, S. (2013). Hydrogen peroxide produced by oral Streptococci induces macrophage cell death. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0062563"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Hernandez-Morfa, M., Olivero, N.B., Zappia, V.E., Pinas, G.E., Reinoso-Vizcaino, N.M., Cian, M.B., Nunez-Fernandez, M., Cortes, P.R., and Echenique, J. (2023). The oxidative stress response of Streptococcus pneumoniae: Its contribution to both extracellular and intracellular survival. Front. Microbiol., 14.","DOI":"10.3389\/fmicb.2023.1269843"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Cocksedge, S.P., Causer, A.J., Winyard, P.G., Jones, A.M., and Bailey, S.J. (2023). Oral Temperature and pH Influence Dietary Nitrate Metabolism in Healthy Adults. Nutrients, 15.","DOI":"10.3390\/nu15030784"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Mazurel, D., Carda-Dieguez, M., Langenburg, T., Ziemyte, M., Johnston, W., Martinez, C.P., Albalat, F., Llena, C., Al-Hebshi, N., and Culshaw, S. (2023). Nitrate and a nitrate-reducing Rothia aeria strain as potential prebiotic or synbiotic treatments for periodontitis. NPJ Biofilms Microbiomes, 9.","DOI":"10.1038\/s41522-023-00406-3"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/13\/2\/279\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T10:36:42Z","timestamp":1759919802000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/13\/2\/279"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1,26]]},"references-count":35,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2025,2]]}},"alternative-id":["microorganisms13020279"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms13020279","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,1,26]]}}}