1887

Journal of Medical Microbiology logo

Volume 62, Issue 1

Maternal oral origin of in adverse pregnancy outcomes as determined using the 16S–23S rRNA gene intergenic transcribed spacer region Free

Abstract

, a common Gram-negative anaerobe prevalent in the oral cavity, possesses the ability to colonize the amniotic cavity and the fetus. However, may also be part of the vaginal microbiota from where it could reach the amniotic tissues. Due to the heterogeneity of , consisting of five subspecies, analysis at the subspecies/strain level is desirable to determine its precise origin. The aims of this study were: (i) to evaluate the use of the 16S–23S rRNA gene intergenic transcribed spacer (ITS) region as a tool to differentiate subspecies of , and (ii) to design a simplified technique based on the ITS to determine the origin of strains associated with adverse pregnancy outcomes. Amplified fragments of the 16S–23S rRNA gene ITS region corresponding to the five subspecies of were subjected to cloning and sequencing to characterize the different ribosomal operons of the subspecies. Distinctive length and sequence patterns with potential to be used for identification of the subspecies/strain were identified. These were used to evaluate the origin of identified in neonatal gastric aspirates (swallowed amniotic fluid) by sequence comparisons with the respective oral and vaginal maternal samples. A simplified technique using a strain-specific primer in a more sensitive nested PCR was subsequently developed to analyse ten paired neonatal–maternal samples. Analysing the variable fragment of the ITS region allowed the identification of subsp. from an oral origin as potentially being involved in neonatal infections. Using a strain-specific primer, the subsp. strain was detected in both neonatal gastric aspirates and maternal oral samples in cases of preterm birth from mothers presenting with localized periodontal pockets. Interestingly, the same strain was not present in the vaginal sample of any case investigated. The 16S–23S rRNA gene ITS can be a useful tool to determine the origin of . The results of this study strongly indicate that subsp. of oral origin could be involved with pregnancy complications.

  • Received:
  • Accepted:
  • Published Online:
© 2013 SGM
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.049452-0
2013-01-01
2024-04-16
Loading full text...

Full text loading...

/deliver/fulltext/jmm/62/1/133.html?itemId=/content/journal/jmm/10.1099/jmm.0.049452-0&mimeType=html&fmt=ahah

References

  1. Bearfield C., Davenport E. S., Sivapathasundaram V., Allaker R. P. 2002; Possible association between amniotic fluid micro-organism infection and microflora in the mouth. BJOG 109:527–533 [View Article][PubMed]
     [Google Scholar]
  2. Benson D. A., Karsch-Mizrachi I., Lipman D. J., Ostell J., Sayers E. W. 2011; GenBank. Nucleic Acids Res 39:Database issueD32–D37 [View Article][PubMed]
     [Google Scholar]
  3. Berres F., Resch G., Ruppert M., Meyer J., Kulik E. M. 2003; [Ribotyping Fusobacterium nucleatum isolates from healthy and diseased periodontium]. Schweiz Monatsschr Zahnmed 113:953–957 (in German) [PubMed]
     [Google Scholar]
  4. Bolstad A. I., Jensen H. B., Bakken V. 1996; Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum. . Clin Microbiol Rev 9:55–71[PubMed]
     [Google Scholar]
  5. Chen T., Yu W. H., Izard J., Baranova O. V., Lakshmanan A., Dewhirst F. E. 2010; The human oral microbiome database: a web accessible resource for investigating oral microbe taxonomic and genomic information. Database (Oxford) 2010:baq013[PubMed] [CrossRef]
     [Google Scholar]
  6. Christensen H., Jørgensen K., Olsen J. E. 1999; Differentiation of Campylobacter coli and C. jejuni by length and DNA sequence of the 16S-23S rRNA internal spacer region. Microbiology 145:99–105 [View Article][PubMed]
     [Google Scholar]
  7. Cole J. R., Chai B., Farris R. J., Wang Q., Kulam-Syed-Mohideen A. S., McGarrell D. M., Bandela A. M., Cardenas E., Garrity G. M., Tiedje J. M. 2007; The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public data. Nucleic Acids Res 35:Database issueD169–D172 [View Article][PubMed]
     [Google Scholar]
  8. Conrads G., Claros M. C., Citron D. M., Tyrrell K. L., Merriam V., Goldstein E. J. 2002; 16S-23S rDNA internal transcribed spacer sequences for analysis of the phylogenetic relationships among species of the genus Fusobacterium. . Int J Syst Evol Microbiol 52:493–499[PubMed]
     [Google Scholar]
  9. Corpet F. 1988; Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890 [View Article][PubMed]
     [Google Scholar]
  10. Dewhirst F. E., Chen T., Izard J., Paster B. J., Tanner A. C., Yu W. H., Lakshmanan A., Wade W. G. 2010; The human oral microbiome. J Bacteriol 192:5002–5017 [View Article][PubMed]
     [Google Scholar]
  11. Dzink J. L., Sheenan M. T., Socransky S. S. 1990; Proposal of three subspecies of Fusobacterium nucleatum Knorr 1922: Fusobacterium nucleatum subsp. nucleatum subsp. nov., comb. nov.; Fusobacterium nucleatum subsp. polymorphum subsp. nov., nom. rev., comb. nov.; and Fusobacterium nucleatum subsp. vincentii subsp. nov., nom. rev., comb. nov. Int J Syst Bacteriol 40:74–78 [View Article][PubMed]
     [Google Scholar]
  12. Gharbia S. E., Shah H. N. 1992; Fusobacterium nucleatum subsp. fusiforme subsp. nov. and Fusobacterium nucleatum subsp. animalis subsp. nov. as additional subspecies within Fusobacterium nucleatum. . Int J Syst Bacteriol 42:296–298 [View Article][PubMed]
     [Google Scholar]
  13. Gharbia S. E., Shah H. N., Lawson P. A., Haapasalo M. 1990; Distribution and frequency of Fusobacterium nucleatum subspecies in the human oral cavity. Oral Microbiol Immunol 5:324–327 [View Article][PubMed]
     [Google Scholar]
  14. Gmür R., Munson M. A., Wade W. G. 2006; Genotypic and phenotypic characterization of fusobacteria from Chinese and European patients with inflammatory periodontal diseases. Syst Appl Microbiol 29:120–130 [View Article][PubMed]
     [Google Scholar]
  15. Goldstein E. J., Summanen P. H., Citron D. M., Rosove M. H., Finegold S. M. 1995; Fatal sepsis due to a β-lactamase-producing strain of Fusobacterium nucleatum subspecies polymorphum . Clin Infect Dis 20:797–800 [View Article][PubMed]
     [Google Scholar]
  16. Gonzales-Marin C., Spratt D. A., Millar M. R., Simmonds M., Kempley S. T., Allaker R. P. 2011; Levels of periodontal pathogens in neonatal gastric aspirates and possible maternal sites of origin. Mol Oral Microbiol 26:277–290 [View Article][PubMed]
     [Google Scholar]
  17. Gonzales-Marin C., Spratt D. A., Millar M. R., Simmonds M., Kempley S. T., Allaker R. P. 2012; Identification of bacteria and potential sources in neonates at risk of infection delivered by caesarean and vaginal birth. J Med Microbiol 61:31–41 [View Article][PubMed]
     [Google Scholar]
  18. Gürtler V., Stanisich V. A. 1996; New approaches to typing and identification of bacteria using the 16S–23S rDNA spacer region. Microbiology 142:3–16 [View Article][PubMed]
     [Google Scholar]
  19. Han Y. W., Shi W., Huang G. T., Kinder Haake S., Park N.-H., Kuramitsu H., Genco R. J. 2000; Interactions between periodontal bacteria and human oral epithelial cells: Fusobacterium nucleatum adheres to and invades epithelial cells. Infect Immun 68:3140–3146 [View Article][PubMed]
     [Google Scholar]
  20. Han Y. W., Redline R. W., Li M., Yin L., Hill G. B., McCormick T. S. 2004; Fusobacterium nucleatum induces premature and term stillbirths in pregnant mice: implication of oral bacteria in preterm birth. Infect Immun 72:2272–2279 [View Article][PubMed]
     [Google Scholar]
  21. Han Y. W., Ikegami A., Bissada N. F., Herbst M., Redline R. W., Ashmead G. G. 2006; Transmission of an uncultivated Bergeyella strain from the oral cavity to amniotic fluid in a case of preterm birth. J Clin Microbiol 44:1475–1483 [View Article][PubMed]
     [Google Scholar]
  22. Han Y. W., Shen T., Chung P., Buhimschi I. A., Buhimschi C. S. 2009; Uncultivated bacteria as etiologic agents of intra-amniotic inflammation leading to preterm birth. J Clin Microbiol 47:38–47 [View Article][PubMed]
     [Google Scholar]
  23. Han Y. W., Fardini Y., Chen C., Iacampo K. G., Peraino V. A., Shamonki J. M., Redline R. W. 2010; Term stillbirth caused by oral Fusobacterium nucleatum . Obstet Gynecol 115:Supplement442–445 [View Article][PubMed]
     [Google Scholar]
  24. Hill G. B. 1998; Preterm birth: associations with genital and possibly oral microflora. Ann Periodontol 3:222–232 [View Article][PubMed]
     [Google Scholar]
  25. Ikegami A., Chung P., Han Y. W. 2009; Complementation of the fadA mutation in Fusobacterium nucleatum demonstrates that the surface-exposed adhesin promotes cellular invasion and placental colonization. Infect Immun 77:3075–3079 [View Article][PubMed]
     [Google Scholar]
  26. Jeffcoat M., Parry S., Sammel M., Clothier B., Catlin A., Macones G. 2011; Periodontal infection and preterm birth: successful periodontal therapy reduces the risk of preterm birth. BJOG 118:250–256 [View Article][PubMed]
     [Google Scholar]
  27. Kapatral V., Anderson I., Ivanova N., Reznik G., Los T., Lykidis A., Bhattacharyya A., Bartman A., Gardner W. other authors 2002; Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586. J Bacteriol 184:2005–2018 [View Article][PubMed]
     [Google Scholar]
  28. Karpathy S. E., Qin X., Gioia J., Jiang H., Liu Y., Petrosino J. F., Yerrapragada S., Fox G. E., Haake S. K. other authors 2007; Genome sequence of Fusobacterium nucleatum subspecies polymorphum – a genetically tractable fusobacterium. PLoS ONE 2:e659 [View Article][PubMed]
     [Google Scholar]
  29. Kim H.-S., Lee D.-S., Chang Y.-H., Kim M. J., Koh S., Kim J., Seong J.-H., Song S. K., Shin H. S. other authors 2010; Application of rpoB and zinc protease gene for use in molecular discrimination of Fusobacterium nucleatum subspecies. J Clin Microbiol 48:545–553 [View Article][PubMed]
     [Google Scholar]
  30. Lawson P. A., Gharbia S. E., Shah H. N., Clark D. R., Collins M. D. 1991; Intrageneric relationships of members of the genus Fusobacterium as determined by reverse transcriptase sequencing of small-subunit rRNA. Int J Syst Bacteriol 41:347–354 [View Article][PubMed]
     [Google Scholar]
  31. Liu H., Redline R. W., Han Y. W. 2007; Fusobacterium nucleatum induces fetal death in mice via stimulation of TLR4-mediated placental inflammatory response. J Immunol 179:2501–2508[PubMed] [CrossRef]
     [Google Scholar]
  32. Miralles R., Hodge R., McParland P. C., Field D. J., Bell S. C., Taylor D. J., Grant W. D., Kotecha S. 2005; Relationship between antenatal inflammation and antenatal infection identified by detection of microbial genes by polymerase chain reaction. Pediatr Res 57:570–577 [View Article][PubMed]
     [Google Scholar]
  33. Nagano Y., Watabe M., Porter K. G., Coulter W. A., Millar B. C., Elborn J. S., Goldsmith C. E., Rooney P. J., Loughrey A., Moore J. E. 2007; Development of a genus-specific PCR assay for the molecular detection, confirmation and identification of Fusobacterium spp. Br J Biomed Sci 64:74–77[PubMed]
     [Google Scholar]
  34. Narayanan S. K., Nagaraja T. G., Chengappa M. M., Stewart G. C. 2001; Electrophoretic mobility anomalies associated with PCR amplification of the intergenic spacer region between 16S and 23S ribosomal RNA genes of Fusobacterium necrophorum . J Microbiol Methods 46:165–169 [View Article][PubMed]
     [Google Scholar]
  35. Offenbacher S., Katz V., Fertik G., Collins J., Boyd D., Maynor G., McKaig R., Beck J. 1996; Periodontal infection as a possible risk factor for preterm low birth weight. J Periodontol 67:Suppl1103–1113[PubMed] [CrossRef]
     [Google Scholar]
  36. Paster B. J., Boches S. K., Galvin J. L., Ericson R. E., Lau C. N., Levanos V. A., Sahasrabudhe A., Dewhirst F. E. 2001; Bacterial diversity in human subgingival plaque. J Bacteriol 183:3770–3783 [View Article][PubMed]
     [Google Scholar]
  37. Patel J. B. 2001; 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. Mol Diagn 6:313–321[PubMed] [CrossRef]
     [Google Scholar]
  38. Popa R., Popa R., Mashall M. J., Nguyen H., Tebo B. M., Brauer S. 2009; Limitations and benefits of ARISA intra-genomic diversity fingerprinting. J Microbiol Methods 78:111–118 [View Article][PubMed]
     [Google Scholar]
  39. Sadeghifard N., Gürtler V., Beer M., Seviour R. J. 2006; The mosaic nature of intergenic 16S–23S rRNA spacer regions suggests rRNA operon copy number variation in Clostridium difficile strains. Appl Environ Microbiol 72:7311–7323 [View Article][PubMed]
     [Google Scholar]
  40. Saito A., Inagaki S., Kimizuka R., Okuda K., Hosaka Y., Nakagawa T., Ishihara K. 2008; Fusobacterium nucleatum enhances invasion of human gingival epithelial and aortic endothelial cells by Porphyromonas gingivalis . FEMS Immunol Med Microbiol 54:349–355 [View Article][PubMed]
     [Google Scholar]
  41. Söller R., Hirsch P., Blohm D., Labrenz M. 2000; Differentiation of newly described antarctic bacterial isolates related to Roseobacter species based on 16S–23S rDNA internal transcribed spacer sequences. Int J Syst Evol Microbiol 50:909–915 [View Article][PubMed]
     [Google Scholar]
  42. Strauss J., White A., Ambrose C., McDonald J., Allen-Vercoe E. 2008; Phenotypic and genotypic analyses of clinical Fusobacterium nucleatum and Fusobacterium periodonticum isolates from the human gut. Anaerobe 14:301–309 [View Article][PubMed]
     [Google Scholar]
  43. Tateishi F., Hasegawa-Nakamura K., Nakamura T., Oogai Y., Komatsuzawa H., Kawamata K., Douchi T., Hatae M., Noguchi K. 2012; Detection of Fusobacterium nucleatum in chorionic tissues of high-risk pregnant women. J Clin Periodontol 39:417–424 [View Article][PubMed]
     [Google Scholar]
  44. Tyrrell G. J., Bethune R. N., Willey B., Low D. E. 1997; Species identification of enterococci via intergenic ribosomal PCR. J Clin Microbiol 35:1054–1060[PubMed]
     [Google Scholar]
  45. Wang G. C., Wang Y. 1997; Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes. Appl Environ Microbiol 63:4645–4650[PubMed]
     [Google Scholar]
  46. Xie H., Gibbons R. J., Hay D. I. 1991; Adhesive properties of strains of Fusobacterium nucleatum of the subspecies nucleatum, vincentii and polymorphum . Oral Microbiol Immunol 6:257–263 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.049452-0
Loading
Maternal oral origin of Fusobacterium nucleatum in adverse pregnancy outcomes as determined using the 16S–23S rRNA gene intergenic transcribed spacer region
J. Med. Microbiol. 62, 133 (2013); https://doi.org/10.1099/jmm.0.049452-0
/content/journal/jmm/10.1099/jmm.0.049452-0
/content/journal/jmm/10.1099/jmm.0.049452-0
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error