Co-Localized or Randomly Distributed? Pair Cross Correlation of In Vivo Grown Subgingival Biofilm Bacteria Quantified by Digital Image Analysis

Claudia Schillinger,Annette Moter,Lawrence E Wolinsky,Judith Kikhney,Ulf B Göbel,Anton Friedmann,Birgit Riep,Annett Petrich,Holger Daims,Renate Lux,Malcolm Gracie Semple

doi:10.1371/journal.pone.0037583

Claudia Schillinger, Annette Moter + Show 9 more

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https://doi.org/10.1371/journal.pone.0037583

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Abstract

The polymicrobial nature of periodontal diseases is reflected by the diversity of phylotypes detected in subgingival plaque and the finding that consortia of suspected pathogens rather than single species are associated with disease development. A number of these microorganisms have been demonstrated in vitro to interact and enhance biofilm integration, survival or even pathogenic features. To examine the in vivo relevance of these proposed interactions, we extended the spatial arrangement analysis tool of the software daime (digital image analysis in microbial ecology). This modification enabled the quantitative analysis of microbial co-localization in images of subgingival biofilm species, where the biomass was confined to fractions of the whole-image area, a situation common for medical samples. Selected representatives of the disease-associated red and orange complexes that were previously suggested to interact with each other in vitro (Tannerella forsythia with Fusobacterium nucleatum and Porphyromonas gingivalis with Prevotella intermedia) were chosen for analysis and labeled with specific fluorescent probes via fluorescence in situ hybridization. Pair cross-correlation analysis of in vivo grown biofilms revealed tight clustering of F. nucleatum/periodonticum and T. forsythia at short distances (up to 6 µm) with a pronounced peak at 1.5 µm. While these results confirmed previous in vitro observations for F. nucleatum and T. forsythia, random spatial distribution was detected between P. gingivalis and P. intermedia in the in vivo samples. In conclusion, we successfully employed spatial arrangement analysis on the single cell level in clinically relevant medical samples and demonstrated the utility of this approach for the in vivo validation of in vitro observations by analyzing statistically relevant numbers of different patients. More importantly, the culture-independent nature of this approach enables similar quantitative analyses for “as-yet-uncultured” phylotypes which cannot be characterized in vitro.

Highlights

Periodontal diseases are prevalent bacterial biofilm infections in humans that involve progressive destruction of the tooth-supporting tissues and tooth loss in the absence of treatment
In addition to species identification, a number of these culture-independent studies implicated novel periodontal pathogens [10,11,12,13,14,15]; (ii) Arrangement of a large panel of cultivable subgingival flora into microbial complexes based on their co-occurrence and association with health and disease [16]: Reflective of the multitude of microorganisms comprising the oral microbiota, clusters of microorganisms rather than single species have been implicated as indicators for periodontal health or disease
Since T. forsythia, a member of the strongly periodontitis-associated red complex, and F. nucleatum of the orange complex physically and synergistically interrelate in vitro [26], we examined the in vivo relevance of their relationship on a quantitative level

Summary

Introduction

Periodontal diseases are prevalent bacterial biofilm infections in humans that involve progressive destruction of the tooth-supporting tissues and tooth loss in the absence of treatment. The so-called ‘‘red complex’’ which is strongly correlated with the severity of disease is comprised of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola These species appear to require the more prevalent ‘‘orange complex’’ species such as Fusobacterium spp. or Prevotella intermedia among others for biofilm integration [17]; (iii) Extensive in vitro examination of the ability of oral species to form aggregates with each other [18,19,20,21,22,23,24,25,26,27]: The multitude of in vitro studies assessing individual interspecies adherence behavior or ‘‘coaggregation’’ allowed a more detailed picture of the elaborate interactions involved in building the architecturally complex oral biofilm networks. Taken together these approaches cumulated in our current understanding that periodontal diseases involve complex synergistic and antagonistic bacterial interactions [34]

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