Necrotizing Gingivitis: Microbial Diversity and Quantification of Protein Secretion in Necrotizing Gingivitis.

Necrotizing gingivitis (NG) is a necrotizing periodontal disease that differs from chronic gingivitis (CG). To date, both the microbiological causes and the involved host cytokine response of NG still remain unclear. Here, we investigated corresponding interdental plaque and serum samples from two groups of Chinese patients with CG (n = 21) or NG (n = 21). The microbiota were studied by 16S rRNA Illumina MiSeq sequencing of the microbial metagenome and by assessing quantitatively the abundance of the phylum Bacteroidetes, the genus Prevotella and the species T. forsythia, P. endodontalis, and P. gingivalis using fluorescence in situ hybridization (FISH). With respect to the associated host response, the levels of 30 inflammatory mediators were quantified by multiplex immunoassay analysis. Differential microbial abundance analysis of the two disease groups revealed at the phylum level that Proteobacteria accounted for 67% of the differentially abundant organisms, followed by organisms of Firmicutes (21%) and Actinobacteria (9%). At the species level, significant differences in abundance were seen for 75 species of which 58 species were significantly more abundant in CG patients. Notably, the FISH analysis revealed that Bacteroidetes was the most prevalent phylum in NG. The multiplex cytokine assay showed significant quantitative differences between the disease groups for eight analytes (GM-CSF, G-CSF, IFN-α, IL-4, IL-13, TNF-α, MIG, and HGF). The G-CSF was found to be the most significantly increased inflammatory protein marker in NG. The next-generation sequencing (NGS) data supported the understanding of NG as a multi-microbial infection with distinct differences to CG in regard to the microbial composition.

Advancement of the 10-species subgingival Zurich biofilm model by examining different nutritional conditions and defining the structure of the in vitro biofilms.

BACKGROUND: Periodontitis is caused by a highly complex consortium of bacteria that establishes as biofilms in subgingival pockets. It is a disease that occurs worldwide and its consequences are a major health concern. Investigations in situ are not possible and the bacterial community varies greatly between patients and even within different loci. Due to the high complexity of the consortium and the availability of samples, a clear definition of the pathogenic bacteria and their mechanisms of pathogenicity are still not available. In the current study we addressed the need of a defined model system by advancing our previously described subgingival biofilm model towards a bacterial composition that reflects the one observed in diseased sites of patients and analysed the structure of these biofilms. RESULTS: We further developed the growth media by systematic variation of key components resulting in improved stability and the firm establishment of spirochetes in the 10-species subgingival Zurich biofilm model. A high concentration of heat-inactivated human serum allowed the best proliferation of the used species. Therefore we further investigated these biofilms by analysing their structure by confocal laser scanning microscopy following fluorescence in situ hybridisation. The species showed mutual interactions as expected from other studies. The abundances of all organisms present in this model were determined by microscopic counting following species-specific identification by both fluorescence in situ hybridisation and immunofluorescence. The newly integrated treponemes were the most abundant organisms. CONCLUSIONS: The use of 50% of heat-inactivated human serum used in the improved growth medium resulted in significantly thicker and more stable biofilms, and the quantitative representation of the used species represents the in vivo community of periodontitis patients much closer than in biofilms grown in the two media with less or no human serum. The appearance of T. denticola, P. gingivalis, and T. forsythia in the top layer of the biofilms, and the high abundance of T. denticola, reflects well the microbial situation observed at diseased sites. The improved model biofilms will allow further investigations of interactions between individual species and of the effects of atmospheric or nutritional changes, as well as interactions with tissue cells.

The phylum Synergistetes in gingivitis and necrotizing ulcerative gingivitis.

The clinical manifestation of necrotizing ulcerative gingivitis (NUG) is distinct from that of common gingivitis in that it is characterized by local necrosis of the gingival tissues, rapid onset, pain and extensive bleeding. The phylum Synergistetes is a novel bacterial phylum consisting of Gram-negative anaerobes, with evidence of presence in biofilms associated with periodontal and endodontic infections. To date, the involvement of members of this phylum in NUG has not been investigated. This study aimed to evaluate the presence and levels of known human oral Synergistetes bacterial clusters in dental plaque from patients with NUG and compare them with those found in gingivitis. Marginal dental plaque samples from 21 NUG and 21 gingivitis patients were analysed quantitatively by fluorescent in situ hybridization and microscopy for members of two oral Synergistetes clusters (A and B) and for Jonquetella anthropi. Synergistetes cluster A bacteria were detected in all samples but at higher levels (9.4-fold) and proportions (2.5-fold) in NUG patients than in gingivitis patients. However, with regard to Synergistetes cluster B bacteria, there were no differences between NUG and gingivitis patients. J. anthropi was detected in only half of the samples and at lower levels than the other taxa. In conclusion, these data demonstrate that Synergistetes cluster A bacteria, but not cluster B bacteria or J. anthropi, are more strongly associated with NUG than with gingivitis.

Subgingival biofilm structure.

Periodontitis is an inflammatory disease of the oral cavity initiated by a microbial biofilm (or 'dental plaque'). Subgingival biofilms in periodontal pockets are not easily analyzed without the loss of structural integrity. These subgingival plaques are structured communities of microorganisms with great phylogenetic diversity embedded in a self-produced extracellular polymeric matrix. For almost three decades, knowledge of the structure of plaque located below the gingival margin has been limited to landmark studies from the 1970s that were unaware of the breadth of microbial diversity we appreciate now. Only recently has technical progress - combining histology, confocal scanning fluorescent microscopy and fluorescent in situ hybridization to localize the most abundant species from different phyla and species associated with periodontitis - provided new insights into the architecture of subgingival biofilms. This review focuses on the structure and composition of subgingival biofilms and discusses current knowledge on the nature of the extracellular matrix. We describe further structural aspects of 'subgingival' biofilms produced in vitro that are gaining considerable interest as we search for models to investigate biofilm development, resistance to antibiotics, extracellular polymeric matrix composition and function, and reciprocal host-cell-to-biofilm interactions.

Validation of the Zürich burn-biofilm model.

BACKGROUND: Despite advances in the use of topical and parenteral antimicrobial therapy and the practice of early tangential burn-wound excision, bacterial infection remains a major problem in the management of burn victims today. The purpose of this study was to design and evaluate a polyspecies biofilm model with bacteria known to cause severe infections in burn patients. The model is simple to prepare, maintain and analyse, and allows for short-term exposure to antimicrobials. RESULTS: Initial experiments showed that it was impossible to establish balanced polyspecies biofilms with an inoculum of Gram-positive and -negative bacteria. After 64.5 h of incubation, the Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) had suppressed the Gram-positives (Enterococcus faecalis, Staphylococcus aureus and Streptococcus intermedius). However, adding the Gram-negative bacteria after 41.5 h to an established biofilm of Gram-positives resulted in a balanced microbial consortium. After 64.5 h, all species were present in high numbers (10(7) to 10(8) colony forming units (CFU) per biofilm). Multiple repetitions showed high reproducibility of biofilm formation without significant differences between and within experiments. Combined fluorescence in situ hybridisation/confocal laser scanning microscopy (FISH/CLSM) analyses, for which biofilms had to be grown on a different non-flexible substrate (hydroxy apatite), revealed that, by 41.5 h, the biofilm consisted of an almost confluent layer of bacteria firmly adherent to the substratum. After 64.5 h (22 h after the addition of the Gram negatives), the biofilm consisted of a confluent mixture of single cells, an abundance of galaxies of bacteria with small lacunae and large amounts of extracellular matrix polysaccharides. CONCLUSIONS: The polyspecies biofilm model contains the most prevalent burn-associated Gram-positive and Gram-negative bacterial pathogens and mimics the Gram-negative shift observed in vivo. It shows excellent reproducibility. It should allow adaptation to the bacterial spectrum prevalent in different burn centres and lead to a much more reliable investigation of the efficiency of topical antimicrobial agents than models operating with planktonic bacteria. The experiments further open up the perspective to create an in vivo model using these biofilms as infectious agents.

Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms.

BACKGROUND: The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella. RESULTS: As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers. CONCLUSIONS: Application of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of oral microbiology, as many of them detect non-oral species and phylogenetic groups of importance in a variety of medical conditions and the food industry.

Targeted delivery of a photosensitizer to Aggregatibacter actinomycetemcomitans biofilm.

The ability to selectively target specific biofilm species with antimicrobials would enable control over biofilm consortium composition, with medical applications in treatment of infections on mucosal surfaces that are colonized by a mixture of beneficial and pathogenic microorganisms. We functionalized a genetically engineered multimeric protein with both a targeting moiety (biotin) and either a fluorophore or a photosensitizer (SnCe6). Biofilm microcolonies of Aggregatibacter actinomycetemcomitans, a periodontal pathogen, were targeted with the multifunctional dodecamer. Streptavidin was used to couple biotinylated dodecamer to a biotinylated anti-A. actinomycetemcomitans antibody. This modular targeting approach enabled us to increase the loading of photosensitizer onto the cells by a cycle of amplification. Scanning laser confocal microscopy was used to characterize transport of fluorescently tagged dodecamer into the microcolonies and targeting of the cells with biotin-labeled, fluorescently tagged dodecamer. Light-induced activity of the targeted photosensitizer reduced the viability of A. actinomycetemcomitans biofilm, as indicated by membrane permeability to propidium iodide. The functionalized multimeric protein promises to be a useful tool for controlling periodontal biofilm consortia and offers a modular design whereby moieties that target different species can be readily combined with the functionalized protein construct.

Oral biofilm architecture on natural teeth.

Periodontitis and caries are infectious diseases of the oral cavity in which oral biofilms play a causative role. Moreover, oral biofilms are widely studied as model systems for bacterial adhesion, biofilm development, and biofilm resistance to antibiotics, due to their widespread presence and accessibility. Despite descriptions of initial plaque formation on the tooth surface, studies on mature plaque and plaque structure below the gum are limited to landmark studies from the 1970s, without appreciating the breadth of microbial diversity in the plaque. We used fluorescent in situ hybridization to localize in vivo the most abundant species from different phyla and species associated with periodontitis on seven embedded teeth obtained from four different subjects. The data showed convincingly the dominance of Actinomyces sp., Tannerella forsythia, Fusobacterium nucleatum, Spirochaetes, and Synergistetes in subgingival plaque. The latter proved to be new with a possibly important role in host-pathogen interaction due to its localization in close proximity to immune cells. The present study identified for the first time in vivo that Lactobacillus sp. are the central cells of bacterial aggregates in subgingival plaque, and that Streptococcus sp. and the yeast Candida albicans form corncob structures in supragingival plaque. Finally, periodontal pathogens colonize already formed biofilms and form microcolonies therein. These in vivo observations on oral biofilms provide a clear vision on biofilm architecture and the spatial distribution of predominant species.

In vitro modeling of host-parasite interactions: the 'subgingival' biofilm challenge of primary human epithelial cells.

BACKGROUND: Microbial biofilms are known to cause an increasing number of chronic inflammatory and infectious conditions. A classical example is chronic periodontal disease, a condition initiated by the subgingival dental plaque biofilm on gingival epithelial tissues. We describe here a new model that permits the examination of interactions between the bacterial biofilm and host cells in general. We use primary human gingival epithelial cells (HGEC) and an in vitro grown biofilm, comprising nine frequently studied and representative subgingival plaque bacteria. RESULTS: We describe the growth of a mature 'subgingival' in vitro biofilm, its composition during development, its ability to adapt to aerobic conditions and how we expose in vitro a HGEC monolayer to this biofilm. Challenging the host derived HGEC with the biofilm invoked apoptosis in the epithelial cells, triggered release of pro-inflammatory cytokines and in parallel induced rapid degradation of the cytokines by biofilm-generated enzymes. CONCLUSION: We developed an experimental in vitro model to study processes taking place in the gingival crevice during the initiation of inflammation. The new model takes into account that the microbial challenge derives from a biofilm community and not from planktonically cultured bacterial strains. It will facilitate easily the introduction of additional host cells such as neutrophils for future biofilm:host cell challenge studies. Our methodology may generate particular interest, as it should be widely applicable to other biofilm-related chronic inflammatory diseases.

Uncultivated Tannerella BU045 and BU063 are slim segmented filamentous rods of high prevalence but low abundance in inflammatory disease-associated dental plaques.

Uncultivated clones BU045 and BU063 and Tannerella forsythia, a 'consensus periodontal pathogen', are the closest known relatives within the genus Tannerella. They have been described to inhabit different ecological niches of the human oral cavity. In this study, fluorescent in situ hybridization (FISH) and immunofluorescence were combined to investigate the prevalence and abundance of BU045 and BU063 in comparison to T. forsythia in plaques from gingivitis, necrotizing ulcerative gingivitis (NUG) and chronic periodontitis. Phylotype-specific FISH probes identified BU045 and BU063 as elongated thin rods with a segmented structure. Two structurally similar and previously unknown, rare phylotypes (127+ and 997+) were also identified due to partial 16S rRNA sequence identity with T. forsythia. In gingivitis, NUG and periodontitis patients, BU045, BU063, 127+, 997+ and T. forsythia were detected with prevalences of 50/83/71/14 and 81%, 100/100/86/17 and 53%, and 100/100/12/0 and 100%, respectively. Supragingivally, colonization density of all five organisms was generally low, rarely exceeding 0.1% of the total biota. In periodontal pocket samples, however, cell numbers of T. forsythia, but not of the uncultivable phylotypes, were greatly elevated. Our data demonstrate that Tannerella phylotypes BU045, BU063, 127+ and 997+ consist of long slim rods with segments, which, with respect to FISH stainability, often behaved as independent units. The phylotypes are frequent but low-level colonizers of various periodontal disease-associated plaques. Their apparent inability to proliferate to high density seems to exclude any relevance for the pathogenesis of periodontal diseases.

A combined immunofluorescence and fluorescent in situ hybridization assay for single cell analyses of dental plaque microorganisms.

A IF-FISH protocol was developed to characterize discrete cell populations within complex dental plaque samples. Optimizing and shortening the 3-step IF procedure and including RNase inhibitor in all IF reagents enabled the combination of both techniques and thus the simultaneous detection and enumeration of bacterial populations with distinct 16S rRNA sequences and/or surface markers of interest.

In vitro quantitative light-induced fluorescence to measure changes in enamel mineralization.

A sensitive, quantitative method for investigating changes in enamel mineralization of specimens subjected to in vitro or in situ experimentation is presented. The fluorescence-detecting instrument integrates a Xenon arc light source and an object positioning stage, which makes it particularly suitable for the nondestructive assessment of demineralized or remineralized enamel. We demonstrate the ability of in vitro quantitative light-induced fluorescence (QLF) to quantify changes in mineralization of bovine enamel discs that had been exposed in vitro to a demineralizing gel (n=36) or biofilm-mediated demineralization challenges (n=10), or were carried in situ by three volunteers during a 10-day experiment (n=12). Further experiments show the technique's value for monitoring the extent of remineralization in 36 specimens exposed in vitro to oral multispecies biofilms and document the repeatability of in vitro QLF measurements (n=10) under standardized assay conditions. The validity of the method is illustrated by comparison with transversal microradiography (TMR), the invasive current gold standard for assessing experimental changes in enamel mineralization. Ten discs with 22 measurement areas for comparison demonstrated a positive correlation between TMR and QLF (r=0.82). Filling a technological gap, this QLF system is a promising tool to assay in vitro nondestructively localized changes in mineralization of enamel specimens.

Genotypic and phenotypic characterization of fusobacteria from Chinese and European patients with inflammatory periodontal diseases.

Phylogenetic and antigenic studies were performed on 48 human oral Fusobacterium strains from Chinese patients with either necrotizing ulcerative gingivitis (NUG) or gingivitis and on 23 Fusobacterium nucleatum or Fusobacterium periodonticum strains from European periodontitis patients. Alignment of partial 16S rRNA gene sequences resulted in a phylogenetic tree that corresponded well with the current classification of oral fusobacteria into F. periodonticum and several subspecies of F. nucleatum, in spite of much minor genetic variability. F. periodonticum, F. nucleatum subsp. animalis and a previously undescribed phylogenetic cluster (C4), that may represent an additional F. nucleatum subspecies, constituted discrete clusters distinct from the remainder of F. nucleatum with high bootstrap values. Chinese and European strains differed markedly with regard to their respective classification patterns, suggesting a predominance of F. peridonticum and F. nucleatum susp. animalis over F. nucleatum subsp. nucleatum and F. nucleatum subsp. fusiforme/vincentii in samples from China. Antigenic typing enabled the association of many previously described serovars with distinct phylogenetic clusters and when applied directly to uncultured clinical samples confirmed the differential distribution of oral Fusobacterium taxa in Chinese and European samples. Bacteria from cluster C4 and F. nucleatum subsp. animalis were significantly more prevalent and accounted for higher cell numbers in NUG than in gingivitis samples, suggesting a possible association of these rarely observed taxa with NUG in Chinese patients.

Application of the Zürich biofilm model to problems of cariology.

The term biofilm is increasingly replacing 'plaque' in the literature, but concepts and existing paradigms are changing much more slowly. There is little doubt that biofilm research will lead to more realistic perception and interpretation of the physiology and pathogenicity of microorganisms colonizing plaques in the oral cavity. There is clear evidence that the genotypic and phenotypic expression profiles of biofilm and planktonic bacteria are different. Several techniques are available today to study multispecies biofilms of oral bacteria, each having its particular advantages and weaknesses. We describe a biofilm model developed in Zürich and demonstrate a number of applications with direct or indirect impact on prophylactic dentistry: spatial arrangement and associative behavior of various species in biofilms; multiplex fluorescent in situ hybridization analysis of oral bacteria in biofilms; use of the biofilm model to predict in vivo efficacy of antimicrobials reliably; mass transport in biofilms; de- and remineralization of enamel exposed to biofilms in vitro. The potential of biofilm experimentation in oral biology has certainly not yet been fully exploited and dozens of possible interesting applications could be investigated. The overall physiological parameters of multispecies biofilms can be measured quite accurately, but it is still impossible to assess in toto the multitude of interactions taking place in such complex systems. What can and should be done is to test hypotheses stemming from experiments with planktonic cells in monospecies cultures. In particular, it will be interesting to investigate the relevance to biofilm composition and metabolism of specific gene products by using appropriate bacterial mutants.

Quantitative detection of Porphyromonas gingivalis fimA genotypes in dental plaque.

We developed quantitative fimA genotype assays and applied them in a pilot study investigating the fimbrial genotype distribution of Porphyromonas gingivalis in European subjects with or without chronic periodontitis. P. gingivalis was found in 71% and 9% of the samples from patients and healthy subjects, respectively. Enumeration of total P. gingivalis cell numbers by polymerase chain reaction and immunofluorescence showed excellent correspondence (r = 0.964). 73% of positive samples contained multiple fimA genotypes, but generally one genotype predominated by one to three orders of magnitude. Genotype II predominated in 60% of the samples. Genotype IV occurred with similar prevalence (73%) as genotype II but predominated in only 20% of the samples. Genotypes I, III and V were of much lower prevalence and cell densities of the latter two remained sparse. Our results suggest marked differences among the fimA genotypes' ability to colonize host sites with high cell numbers.

Gingival crevice microbiota from Chinese patients with gingivitis or necrotizing ulcerative gingivitis.

The objective of this study was to quantitatively compare the bacterial population structure in plaque from the gingival margin of two groups of 21 Chinese patients with gingivitis or necrotizing ulcerative gingivitis (NUG). Subjects were recruited in four dental clinics in Eastern China. Samples were quantitatively assessed by immunofluorescence and fluorescent in situ hybridization for taxa known to be associated with periodontal diseases. The analyses showed that the fusiform taxa (Fusobacterium nucleatum/Fusobacterium periodonticum, Leptotrichia buccalis, Tannerella forsythensis, and Capnocytophaga sp.), Campylobacter rectus, Prevotella intermedia, Prevotella nigrescens, Selenomonas sputigena, and treponemes were present in both groups with high prevalence. Porphyromonas gingivalis and Actinomyces gerencseriae were much more prevalent in the NUG group. Quantitatively, most taxa, including P. gingivalis, F. nucleatum and the treponemes, accounted, on average, for < 3% of the total bacterial cell number. Only P. intermedia/P. nigrescens, P. gingivalis, S. sputigena, A. gerencseriae, and the sum of all monitored suspected periodontal pathogens were significantly increased in the NUG group. The present study demonstrates for both groups a highly diverse plaque composition and suggests that, etiologically, the overall concentration and the concerted effects of the entire group of opportunistic pathogens thriving in NUG-associated plaque are of prime importance.

Mass transport of macromolecules within an in vitro model of supragingival plaque.

The aim of this study was to examine the diffusion of macromolecules through an in vitro biofilm model of supragingival plaque. Polyspecies biofilms containing Actinomyces naeslundii, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus sobrinus, Veillonella dispar, and Candida albicans were formed on sintered hydroxyapatite disks and then incubated at room temperature for defined periods with fluorescent markers with molecular weights ranging from 3,000 to 900,000. Subsequent examination by confocal laser scanning microscopy revealed that the mean square penetration depths for all tested macromolecules except immunoglobulin M increased linearly with time, diffusion coefficients being linearly proportional to the cube roots of the molecular weights of the probes (range, 10,000 to 240,000). Compared to diffusion in bulk water, diffusion in the biofilms was markedly slower. The rate of diffusion for each probe appeared to be constant and not a function of biofilm depth. Analysis of diffusion phenomena through the biofilms suggested tortuosity as the most probable explanation for retarded diffusion. Selective binding of probes to receptors present in the biofilms could not explain the observed extent of retardation of diffusion. These results are relevant to oral health, as selective attenuated diffusion of fermentable carbohydrates and acids produced within dental plaque is thought to be essential for the development of carious lesions.

Direct quantitative differentiation between Prevotella intermedia and Prevotella nigrescens in clinical specimens.

This paper describes a quantitative fluorescent in situ hybridization (FISH) assay for the differential identification of Prevotella intermedia and Prevotella nigrescens in clinical samples, and compares its performance with less discriminatory culture and quantitative immunofluorescence (IF) assays. Fluorescence-labelled oligonucleotide probes directed to specific 16S rRNA sequences of P. intermedia, P. nigrescens, Prevotella pallens and Prevotella denticola were hybridized under stringent conditions with cultured reference strains or plaque samples from deep periodontal pockets. Probe specificity was defined with strains from multiple oral Prevotella species. The lower detection level of the assays was approximately 3x10(3) target cells per ml of plaque-sample suspension. P. intermedia, P. nigrescens, P. pallens and P. denticola were detected in plaques with prevalences of 69, 67, 0 and 28%, respectively. On average, 3.9 x 10(6) P. intermedia, 3.1 x 10(6) P. nigrescens and 5.6 x 10(5) P. denticola cells were counted per positive sample. All three species were found almost exclusively in dense mixed aggregates. Quantitative FISH data agreed satisfactorily with corresponding IF data (r=0.711). Both FISH and IF enumerations of the sum of P. intermedia and P. nigrescens markedly exceeded the c.f.u. counts of black-pigmented colonies in Porphyromonas gingivalis-free cultured subgingival plaques. The results demonstrate the validity of this new assay. Unlike established IF, culture, PCR or checkerboard DNA hybridization assays, this FISH assay differentiates quantitatively between P. intermedia and P. nigrescens, provides visual accuracy control, and offers insights into the spatial distribution of the target cells within a clinical sample.