Analysis of the oral bacterial flora in subjects with periodontal diseases

A bacterioscopic study of the bacterial flora of 15 healthy and 15 periodontal disease carriers is presented in relation to the possibly pathogenetic role of oral bacterial flora in the development of human periodontal disease. Study samples taken from sulcus and gingival pockets were examined by darkfield microscopy. Results show that bacterial flora from periodontal disease subjects differs from that in healthy subjects in that spirochetes are present and the incidence of straight and motile rods is higher than of coccoid cells.     

Actinobacillus (Haemophilus) actinomycetemcomitans in periodontal disease

Actinobacillus (Haemophilus) actinomycetemcomitans is a facultatively anaerobic, gram-negative coccobacillus which is a possible etiological agent in juvenile periodontitis (JP). In this study, bacterial flora, especially the occurrence of A. actinomycetemcomitans, in the periodontal pockets of one juvenile with gingivitis (G), one JP patients, five rapidly progressive periodontitis (RP) patients and one adult periodontitis(AP) patient, and one adult with healthy periodontium was investigated using a blood agar medium and a selective medium for A. actinomycetemcomitans. Eight hundred and sixty-five bacteria were isolated from the periodontal pockets, examined for their gram-stain, cell morphologies, relations to O2 and CO2 and catalase reaction, and divided into 21 groups on the basis of these characters. Among the isolates, 604 isolates were further characterized biochemically and identified. A. actinomycetemcomitans was found as 0.2% of the flora of a site in the JP patient, as 9% of the flora of a site in the G patient, and as 19% and 1%, respectively, of the flora of a site in the two RP patients. However, the organism was not detected in another lesion site of the JP patient. In our JP and RP patients, Fusobacterium, Wolinella, Streptococcus, and obligately anaerobic, gram-positive cocci were frequently found at high levels. The bacterial flora of the G and AP patients were more heterogeneous and included Bacteroides at relatively high proportions. These results indicate that A. actinomycetemcomitans is not always associated with JP but occurred in some patients with RP and G.     

Pregnancy and periodontal tissues

Periodontitis is today considered to be a serious disease of periodontal tissues, one caused in most cases by bacterial infection which stimulates proteolysis and osteolysis of the tissues. Typical for the disease is formation of periodontal pockets and a chronic destructive inflammation which impacts on the whole organism. Periodontopathic bacteria colonized in a subgingival biofilm cannot be removed by common oral hygiene. Overproduction of bacteria and other pro-inflammatory mediators can increase the total pro-inflammatory state of the organism in pregnant women. Increased levels of some pro-inflammatory cytokines (PGE2) and cells in fetoplacental space can lead to premature rupture of membranes and subsequent delivery of immature babies. An increasing number of studies in this field provide evidence that good professional care and personal oral hygiene can bring benefits through a decreased prevalence of preterm low birth weight infants (PLBWI) in women suffering periodontitis, although definitive conclusions have not yet been reached. Future mothers with periodontitis can run not only an increased risk of PLWBI but often also suffer pre-eclampsia - a state called acute atherosis - which can be ethiopathogenetically associated with high concentrations of various pro-inflammatory mediators. An increased production of female hormones during pregnancy contributes to the development of gingivitis and periodontitis because vascular permeability and possible tissue edema are both increased.     

In situ morphology of the gut microbiota of wood-eating termites [Reticulitermes flavipes (Kollar) and Coptotermes formosanus Shiraki].

Light microscopy and scanning and transmission electron microscopy were used to examine the in situ morphology of the gut microbiota of Reticulitermes flavipes and Caoptotermes formosanus. Laboratory-maintained termites were used and, for R. flavipes, specimens were also prepared immediately after collection from a natural infestation. The latter endeavor enabled a study of different castes and developmental stages of R. flavipes and revealed differences in the microbiota of field versus laboratory specimens. The termite paunch microbiota consisted of an abundance of morphologically diverse bacteria and protozoa. Thirteen bacterial morphotypes in the paunch were described in detail: seven were observed only in R. flavipes, three were observed only in C. formosanus, and three were common to both termite species. The paunch epithelium was densely colonized by bacteria, many of which possessed holdfast elements that secured them tightly to this tissue and to other bacterial cells. Besides bacteria, the protozoan Pyrsonympha vertens adhered to the paunch epithelium of R. flavipes by means of an attachment organelle. Cuplike indentations were present on the paunch epithelial surface and were sites of bacterial aggregation. Ultrastructural features of cups suggested their involvement in ion absorption. In addition to the paunch, the midgut was also colonized by bacteria that were situated between epithelial microvilli. Results suggest that bacteria are an integral part of the gut ecosystem.     

<Emphasis Type="Italic">Filifactor alocis</Emphasis> - involvement in periodontal biofilms

Background  

Bacteria in periodontal pockets develop complex sessile communities that attach to the tooth surface. These highly dynamic microfloral environments challenge both clinicians and researchers alike. The exploration of structural organisation and bacterial interactions within these biofilms is critically important for a thorough understanding of periodontal disease. In recent years, Filifactor alocis, a fastidious, Gram-positive, obligately anaerobic rod was repeatedly identified in periodontal lesions using DNA-based methods. It has been suggested to be a marker for periodontal deterioration. The present study investigated the epidemiology of F. alocis in periodontal pockets and analysed the spatial arrangement and architectural role of the organism in in vivo grown subgingival biofilms.     

Identification of Intracellular Bacteria in the Ciliate Balantidium ctenopharyngodoni (Ciliophora,Litostomatea)

The ciliate Balantidium ctenopharyngodoni is the most prominent protist in the guts of grass carp, where it mainly inhabits the creamy luminal contents of the hindgut. Ciliates are generally colonized by microorganisms via phagotrophic feeding. In order to study the intracellular bacteria in this ciliate, we have successfully established it in in vitro culture. Herein, we investigated and compared the bacterial community structures of cultured and freshly collected B. ctenopharyngodoni. The results showed that these two groups exhibited different bacterial communities. The most abundant bacterial family in freshly collected samples was Enterobacteriaceae, while in cultured samples it was Fusobacteriaceae. In addition, a key intracellular bacterium, Cetobacterium somerae, was identified in the cytoplasm of cultured ciliates using fluorescence in situ hybridization (FISH). This study shows that ciliates can retain the intracellular bacteria acquired in the natural habitat for quite a long time, but the bacterial community structure of ciliates eventually changes after a long period of cultivation.     

Microscope-Based Imaging Platform for Large-Scale Analysis of Oral Biofilms

A microscopic method for noninvasively monitoring oral biofilms at the macroscale was developed to describe the spatial distribution of biofilms of different bacterial composition on bovine enamel surfaces (BES). For this purpose, oral biofilm was grown in situ on BES that were fixed at approximal sites of individual upper jaw acrylic devices worn by a volunteer for 3 or 5 days. Eubacteria, Streptococcus spp., and Fusobacterium nucleatum were stained using specific fluorescence in situ hybridization (FISH) probes. The resulting fluorescence signals were subsequently tested by confocal laser scanning microscopy (CLSM) and monitored by an automated wide-field microscope-based imaging platform (Scan∧R). Automated image processing and data analysis were conducted by microscope-associated software and followed by statistical evaluation of the results. The full segmentation of biofilm images revealed a random distribution of bacteria across the entire area of the enamel surfaces examined. Significant differences in the composition of the microflora were recorded across individual as well as between different enamel surfaces varying from sparsely colonized (47.26%) after 3 days to almost full surface coverage (84.45%) after 5 days. The enamel plates that were positioned at the back or in the middle of the oral cavity were found to be more suitable for the examination of biofilms up to 3 days old. In conclusion, automated microscopy combined with the use of FISH can enable the efficient visualization and meaningful quantification of bacterial composition over the entire sample surface. Due to the possibility of automation, Scan∧R overcomes the technical limitations of conventional CLSM.     

Colonization of barley (Hordeum vulgare) with Salmonella enterica and Listeria spp

Colonization of barley plants by the food-borne pathogens Salmonella enterica serovar typhimurium and three Listeria spp. (L. monocytogenes, L. ivanovii, L. innocua) was investigated in a monoxenic system. Herbaspirillum sp. N3 was used as a positive control and Escherichia coli HB101 as a negative control for endophytic root colonization. Colonization of the plants was tested 1-4 weeks after inoculation by determination of CFU, specific PCR assays and fluorescence in situ hybridization (FISH) with fluorescently labelled oligonucleotide probes in combination with confocal laser scanning microscopy (CLSM). Both S. enterica strains were found as endophytic colonizers of barley roots and reached up to 2.3 x 10(6) CFU per g root fresh weight after surface sterilization. The three Listeria strains had 10-fold fewer cell numbers after surface sterilization on the roots and therefore were similar to the results of nonendophytic colonizers, such as E. coli HB101. The FISH/CSLM approach demonstrated not only high-density colonization of the root hairs and the root surface by S. enterica but also a spreading to subjacent rhizodermis layers and the inner root cortex. By contrast, the inoculated Listeria spp. colonized the root hair zone but did not colonize other parts of the root surface. Endophytic colonization of Listeria spp. was not observed. Finally, a systemic spreading of S. enterica to the plant shoot (stems and leaves) was demonstrated using a specific PCR analysis and plate count technique.     

Microbial composition and structure of aerobic granular sewage biofilms

Aerobic activated sludge granules are dense, spherical biofilms which can strongly improve purification efficiency and sludge settling in wastewater treatment processes. In this study, the structure and development of different granule types were analyzed. Biofilm samples originated from lab-scale sequencing batch reactors which were operated with malthouse, brewery, and artificial wastewater. Scanning electron microscopy, light microscopy, and confocal laser scanning microscopy together with fluorescence in situ hybridization (FISH) allowed insights into the structure of these biofilms. Microscopic observation revealed that granules consist of bacteria, extracellular polymeric substances (EPS), protozoa and, in some cases, fungi. The biofilm development, starting from an activated sludge floc up to a mature granule, follows three phases. During phase 1, stalked ciliated protozoa of the subclass Peritrichia, e.g., Epistylis spp., settle on activated sludge flocs and build tree-like colonies. The stalks are subsequently colonized by bacteria. During phase 2, the ciliates become completely overgrown by bacteria and die. Thereby, the cellular remnants of ciliates act like a backbone for granule formation. During phase 3, smooth, compact granules are formed which serve as a new substratum for unstalked ciliate swarmers settling on granule surfaces. These mature granules comprise a dense core zone containing bacterial cells and EPS and a loosely structured fringe zone consisting of either ciliates and bacteria or fungi and bacteria. Since granules can grow to a size of up to several millimeters in diameter, we developed and applied a modified FISH protocol for the study of cryosectioned biofilms. This protocol allows the simultaneous detection of bacteria, ciliates, and fungi in and on granules.     

Black-pigmented Gram-negative anaerobes in periodontitis

Abstract Black-pigmented Gram-negative anaerobes have been associated with periodontal disease and tooth loss since they were first isolated by Burdon in 1928. Porphyromonas gingivalis , which is usually not isolated from children, adolescents or adults with no periodontal breakdown, has been recognized as one of the most important periodontopathogens. Its presence is strongly correlated with deep periodontal pockets, which are assumed to be its main habitat. Correlations have been shown also with attachment loss, clinical inflammation and serum antibody levels, indicating an aetiological role in the periodontal disease. Their pathogenicity in animal models resembling periodontal disease is documented. They are frequently isolated from periodontal abscesses. The relationship between Prevotella intermedia and periodontal disease is not clear. It is frequently isolated from advanced periodontitis, often as the only black-pigmented Gram-negative anaerobic species; however, the prevalence in adults with no periodontal breakdown is high. It is found frequently in periodontal abscesses and in acute necrotizing and ulcerative gingivitis. Serogroup I is found predominantly in deep periodontal pockets, whereas all serogroups (I–III) are found in shallow pockets and gingivitis. No conclusive difference in pathogenicity between serogroups has been found. Pr. melaninogenica, Pr. denticola and Pr. loescheii are frequently found in the gingival crevice in preschool children and other age groups with gingivitis, but are seldom found in deep periodontal pockets.     

Comparative study of a new quantitative real-time PCR targeting the xylulose-5-phosphate/fructose-6-phosphate phosphoketolase bifidobacterial gene (xfp) in faecal samples with two fluorescence in situ hybridization methods

Aims:  To detect and enumerate bifidobacteria in faeces with a new quantitative multiplex real-time PCR (qPCR) method and to compare the results obtained with fluorescence in situ hybridization (FISH) methods.
Methods and Results:  A multiplex qPCR assay was developed, which enabled the enumeration of Bifidobacterium spp. by targeting the bifidobacterial xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene ( xfp ) and total bacteria using universal Eub-primers targeting 16S rRNA gene from the domain bacteria. The qPCR assay showed high sensitivity and specificity and a low detection limit of about 2·5 × 10³ bifidobacterial cells per gram of faeces. The qPCR results were compared with FISH combined with microscopy or flow cytometry (FCM). No statistical differences among bifidobacterial counts averages measured in adult faeces with the three methods were observed. Total bacterial count averages were higher with the FISH method coupled with microscopic analyses compared to FISH with FCM, whereas total cell numbers estimated by qPCR were intermediate between the two FISH methods.
Conclusions:  The new qPCR assay was shown to be sensitive, rapid and accurate for enumerating bifidobacteria in faeces.
Significance and Impact of the Study:  This method is a valuable alternative for other molecular methods for detecting faecal bifidobacteria, especially when their counts are below the detection limit of the FISH methods.     

A Biofilm Pocket Model to Evaluate Different Non-Surgical Periodontal Treatment Modalities in Terms of Biofilm Removal and Reformation,Surface Alterations and Attachment of Periodontal Ligament Fibroblasts

Background and Aim

There is a lack of suitable in vitro models to evaluate various treatment modalities intending to remove subgingival bacterial biofilm. Consequently, the aims of this in vitro-study were: a) to establish a pocket model enabling mechanical removal of biofilm and b) to evaluate repeated non-surgical periodontal treatment with respect to biofilm removal and reformation, surface alterations, tooth hard-substance-loss, and attachment of periodontal ligament (PDL) fibroblasts.

Material and Methods

Standardized human dentin specimens were colonized by multi-species biofilms for 3.5 days and subsequently placed into artificially created pockets. Non-surgical periodontal treatment was performed as follows: a) hand-instrumentation with curettes (CUR), b) ultrasonication (US), c) subgingival air-polishing using erythritol (EAP) and d) subgingival air-polishing using erythritol combined with chlorhexidine digluconate (EAP-CHX). The reduction and recolonization of bacterial counts, surface roughness (Ra and Rz), the caused tooth substance-loss (thickness) as well as the attachment of PDL fibroblasts were evaluated and statistically analyzed by means of ANOVA with Post-Hoc LSD.

Results

After 5 treatments, bacterial reduction in biofilms was highest when applying EAP-CHX (4 log10). The lowest reduction was found after CUR (2 log10). Additionally, substance-loss was the highest when using CUR (128±40 µm) in comparison with US (14±12 µm), EAP (6±7 µm) and EAP-CHX (11±10) µm). Surface was roughened when using CUR and US. Surfaces exposed to US and to EAP attracted the highest numbers of PDL fibroblasts.

Conclusion

The established biofilm model simulating a periodontal pocket combined with interchangeable placements of test specimens with multi-species biofilms enables the evaluation of different non-surgical treatment modalities on biofilm removal and surface alterations. Compared to hand instrumentation the application of ultrasonication and of air-polishing with erythritol prevents from substance-loss and results in a smooth surface with nearly no residual biofilm that promotes the reattachment of PDL fibroblasts.     

Biodegradable dental implants of ciprofloxacin beta-cyclodextrin inclusion complex in the treatment of periodontitis

Dental implants of ciprofloxacin beta-cyclodextrin inclusion complex were formulated using poly (epsilon-caprolactone), a biodegradable polymer and evaluated. Clinical evaluation was carried out in ten patients with acute peridontitis. Various clinical parameters, viz. gingival index, plaque score, attachment gain, reduction in pocket depth were evaluated at 10, 20, 30, 40 days of treatment and compared with placebo as control. A significant (P < 0.0001) improvement in the healing of periodontal pockets treated with ciprofloxacin beta-cyclodextrin implant was observed in most of the clinical parameters. Estimation of gingival crevicular fluids (GCF) for the drug content revealed that drug levels above the minimum inhibitory concentration (10.2 micrograms/mg) for many of the periodontal pathogens were maintained throughout the period of study (40 days). This confirms the clinical efficacy of the dose and the duration of the study. It was found that biodegradable carrier was better accepted than the non-biodegradable carriers reported earlier.     

Direct image-based correlative microscopy technique for coupling identification and structural investigation of bacterial symbionts associated with metazoans

Coupling prokaryote identification with ultrastructural investigation of bacterial communities has proven difficult in environmental samples. Prokaryotes can be identified by using specific probes and fluorescence in situ hybridization (FISH), but resolution achieved by light microscopes does not allow ultrastructural investigation. In the case of symbioses involving bacteria associated with metazoan tissues, FISH-based studies often indicate the co-occurrence of several bacterial types within a single host species. The ultrastructure is then relevant to address host and bacterial morphology and the intra- or extracellular localization of symbionts. A simple protocol for correlative light and electron microscopy (CLEM) is presented here which allows FISH-based identification of specific 16S rRNA phylotypes and transmission electron microscopy to be performed on a same sample. Image analysis tools are provided to superimpose images obtained and generate overlays. This procedure has been applied to two symbiont-bearing metazoans, namely, aphids and deep-sea mussels. The FISH protocol was modified to take into account constraints associated with the use of electron microscopy grids, and intense and specific signals were obtained. FISH signals were successfully overlaid with bacterial morphotypes in aphids. We thus used the method to address the question of symbiont morphology and localization in a deep-sea mussel. Signals from a type I methanotroph-related phylotype were associated with morphotypes displaying the stacked internal membranes typical for this group and three-dimensional electron tomography was performed, confirming for the first time the correspondence between morphology and phylotype. CLEM is thus feasible and reliable and could emerge as a potent tool for the study of prokaryotic communities.     

What determines nasal carriage of Staphylococcus aureus?

Nasal carriage of Staphylococcus aureus is an important risk factor for infection by this organism in both community and hospital settings; this article reviews the role of host and bacterial factors in carriage. A host genetic influence appears likely but the phenotypic determinants are unknown. Possibilities include variability in host adhesins, immune response or secretion of antimicrobial molecules. Colonization resistance by S. aureus, together with the observation that persistent carriers often carry a single strain whereas intermittent carriers can be colonized with unrelated strains over time, suggests that bacterial factors could also be involved.     

Studies on the possible application of molecular methods in diagnosing carriers and in similarity analysis of group B streptococci (Streptococcus agalactiae)

The most popular method of GBS identification in Poland currently is by culturing on enriched agar and verifying the Lancefield Group using special latex agglutination kits. However, the classical methods are time-consuming and their sensitivity is insufficient therefore it is becoming more common to try and apply molecular methods which are characterized by high sensitivity and rapid results. Moreover, molecular methods give us the possibility to carry out epidemiological investigations and gene detection, for instance for antibiotic resistance. It was confirmed that PCR and FISH procedures may be effective in rapid detection of GBS. Thanks to RAPD methods we showed that newborns born to colonized mothers were colonized by GBS strains which originated from the mother, irrespective of the way and the course of labour. Additionally, we detected GBS colonization in children who were born to mothers who were not colonized by GBS. These children were probably colonized with strains coming from hospital environment. More studies are needed to elucidate the route of transmission and the role of colonization of the medical staff. Using multiplex PCR we showed the presence of ermA, ermB and ermC genes in phenotypically confirmed MLS, GBS strains.     

Enigmatic dual symbiosis in the excretory organ of Nautilus macromphalus (Cephalopoda: Nautiloidea)

Symbiosis is an important driving force in metazoan evolution and the study of ancient lineages can provide an insight into the influence of symbiotic associations on morphological and physiological adaptations. In the 'living fossil' Nautilus, bacterial associations are found in the highly specialized pericardial appendage. This organ is responsible for most of the excretory processes (ultrafiltration, reabsorption and secretion) and secretes an acidic ammonia-rich excretory fluid. In this study, we show that Nautilus macromphalus pericardial appendages harbour a high density of a beta-proteobacterium and a coccoid spirochaete using transmission electron microscopy, comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH). These two bacterial phylotypes are phylogenetically distant from any known bacteria, with ammonia-oxidizing bacteria as the closest relatives of the beta-proteobacterium (above or equal to 87.5% sequence similarity) and marine Spirochaeta species as the closest relatives of the spirochaete (above or equal to 89.8% sequence similarity), and appear to be specific to Nautilus. FISH analyses showed that the symbionts occur in the baso-medial region of the pericardial villi where ultrafiltration and reabsorption processes take place, suggesting a symbiotic contribution to the excretory metabolism.     

What determines nasal carriage of Staphylococcus aureus?

Nasal carriage of Staphylococcus aureus is an important risk factor for infection by this organism in both community and hospital settings; this article reviews the role of host and bacterial factors in carriage. A host genetic influence appears likely but the phenotypic determinants are unknown. Possibilities include variability in host adhesins, immune response or secretion of antimicrobial molecules. Colonization resistance by S. aureus, together with the observation that persistent carriers often carry a single strain whereas intermittent carriers can be colonized with unrelated strains over time, suggests that bacterial factors could also be involved.     

Colonization of mucin by human intestinal bacteria and establishment of biofilm communities in a two-stage continuous culture system

The human large intestine is covered with a protective mucus coating, which is heavily colonized by complex bacterial populations that are distinct from those in the gut lumen. Little is known of the composition and metabolic activities of these biofilms, although they are likely to play an important role in mucus breakdown. The aims of this study were to determine how intestinal bacteria colonize mucus and to study physiologic and enzymatic factors involved in the destruction of this glycoprotein. Colonization of mucin gels by fecal bacteria was studied in vitro, using a two-stage continuous culture system, simulating conditions of nutrient availability and limitation characteristic of the proximal (vessel 1) and distal (vessel 2) colon. The establishment of bacterial communities in mucin gels was investigated by selective culture methods, scanning electron microscopy, and confocal laser scanning microscopy, in association with fluorescently labeled 16S rRNA oligonucleotide probes. Gel samples were also taken for analysis of mucin-degrading enzymes and measurements of residual mucin sugars. Mucin gels were rapidly colonized by heterogeneous bacterial populations, especially members of the Bacteroides fragilis group, enterobacteria, and clostridia. Intestinal bacterial populations growing on mucin surfaces were shown to be phylogenetically and metabolically distinct from their planktonic counterparts.     

Colonization of Mucin by Human Intestinal Bacteria and Establishment of Biofilm Communities in a Two-Stage Continuous Culture System

The human large intestine is covered with a protective mucus coating, which is heavily colonized by complex bacterial populations that are distinct from those in the gut lumen. Little is known of the composition and metabolic activities of these biofilms, although they are likely to play an important role in mucus breakdown. The aims of this study were to determine how intestinal bacteria colonize mucus and to study physiologic and enzymatic factors involved in the destruction of this glycoprotein. Colonization of mucin gels by fecal bacteria was studied in vitro, using a two-stage continuous culture system, simulating conditions of nutrient availability and limitation characteristic of the proximal (vessel 1) and distal (vessel 2) colon. The establishment of bacterial communities in mucin gels was investigated by selective culture methods, scanning electron microscopy, and confocal laser scanning microscopy, in association with fluorescently labeled 16S rRNA oligonucleotide probes. Gel samples were also taken for analysis of mucin-degrading enzymes and measurements of residual mucin sugars. Mucin gels were rapidly colonized by heterogeneous bacterial populations, especially members of the Bacteroides fragilis group, enterobacteria, and clostridia. Intestinal bacterial populations growing on mucin surfaces were shown to be phylogenetically and metabolically distinct from their planktonic counterparts.