The purpose of this study was to analyze the bacterial diversity in persistent apical lesions on root-filled teeth by using culture-independent molecular methods.
Twenty surgically removed apical lesions from therapy-resistant teeth were examined for the presence of bacterial DNA using PCR targeting the 16s ribosomal RNA gene, followed by cloning and sequencing.
Bacterial DNA was detected in 17 of the 20 samples (85%). A total of 236 clones were analyzed. Seven different bacterial phyla were represented and a total of 75 different bacterial taxa were identified; 36% of the species have not yet been cultivated. Commonly detected bacterial species included Fusobacterium spp., Prevotella spp., Tannerella forsythia, Porphyromonas endodontalis, Treponema denticola, Bacteroidetes spp., Peptostreptococcus spp., and Streptococcus spp.
A wide range of bacteria was identified in periapical lesions on therapy-resistant teeth. These bacteria may contribute in the etiology of periapical infection and impede healing of these lesions.
bacterial phyla; endodontic infection; therapy-resistant teeth; 16s ribosomal RNA; sequencing
Bacterial biofilms have been found to develop on root surfaces outside the apical foramen and be associated with refractory periapical periodontitis. However, it is unknown which bacterial species form extraradicular biofilms. The present study aimed to investigate the identity and localization of bacteria in human extraradicular biofilms. Twenty extraradicular biofilms, used to identify bacteria using a PCR-based 16S rRNA gene assay, and seven root-tips, used to observe immunohistochemical localization of three selected bacterial species, were taken from 27 patients with refractory periapical periodontitis. Bacterial DNA was detected from 14 of the 20 samples, and 113 bacterial species were isolated. Fusobacterium nucleatum (14 of 14), Porphyromonas gingivalis (12 of 14), and Tannellera forsythensis (8 of 14) were frequently detected. Unidentified and uncultured bacterial DNA was also detected in 11 of the 14 samples in which DNA was detected. In the biofilms, P. gingivalis was immunohistochemically detected in all parts of the extraradicular biofilms. Positive reactions to anti-F. nucleatum and anti-T. forsythensis sera were found at specific portions of the biofilm. These findings suggested that P. gingivalis, T. forsythensis, and F. nucleatum were associated with extraradicular biofilm formation and refractory periapical periodontitis.
We evaluated the usefulness of PCR analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) and the CTX-M extended-spectrum β-lactamase (ESBL) followed by microchip gel electrophoresis (MGE) for direct identification and CTX-M detection of Gram-negative bacteria (GNB) from positive blood culture bottles. Of 251 GNB isolated from blood cultures containing a single bacterium, 225 (90%) were correctly identified at the species level directly from positive blood culture bottles by comparing the ITS-PCR patterns of the sample strain with those of the control strains. There were no cases of incorrect identification. Limitations encountered included the inability to detect mixed cultures (four bottles) as well as some species (Enterobacter species and Klebsiella oxytoca) demonstrating identical ITS-PCR patterns. A total of 109 ESBL-producing isolates from various clinical materials obtained between January 2005 and December 2008 were examined for blaCTX-M, blaSHV, and blaTEM genes by PCR and sequences of PCR products. CTX-M ESBL was detected in 105 isolates, and SHV ESBL was detected in two isolates. The remaining two isolates (K. oxytoca) were shown to harbor blaOXY. Twenty (19%) of 104 Escherichia coli isolates from blood cultures were suspected to produce ESBL by the combination disk method, and these isolates were shown to harbor CTX-M ESBL by PCR-MGE. The results were obtained within 1.5 h at a calculated cost of $6.50 per specimen. In conclusion, simultaneous detection of ITS length polymorphisms and blaCTX-M by single PCR followed by MGE is useful for rapid, cost-effective, and reliable species-level identification of CTX-M ESBL-producing GNB responsible for bloodstream infections.
PCR-single-strand conformation polymorphism (PCR-SSCP) analysis is a rapid and convenient technique for the detection of mutations and allelic variants. We have adapted this technique for the identification of bacteria by PCR with fluorescein-labeled primers chosen from the conserved regions of the 16S rRNA gene flanking a variable region. The PCR product was denatured, separated on a nondenaturing gel, and detected by an automated DNA sequencer. The mobility of the single-stranded DNA is sequence dependent and allows the identification of a broad panel of bacteria. A single nucleotide difference in the amplified region was sufficient to obtain different PCR-SSCP patterns. The simultaneous amplification of multiple polymorphic regions by multiplex PCR with subsequent multiplex SSCP increased the discriminatory power of PCR-SSCP. A broad range of gram-negative and gram-positive bacteria were tested by PCR-SSCP, including, e.g., Escherichia coli, Enterobacter spp., Klebsiella spp., Haemophilus spp., Neisseria spp., Staphylococcus spp, Streptococcus spp., Enterococcus spp., and Bacillus spp. In total, a panel of 178 strains of bacteria representing 51 species in 21 genera was examined. Although a limited number of strains from each species were tested, the strains tested gave species-specific patterns, with only one exception: Shigella species were indistinguishable from E. coli. PCR is a sensitive technique; as few as 10 CFU of E. coli was sufficient to produce PCR-SSCP patterns suitable for identification. The whole fluorescence PCR-SSCP procedure takes approximately 8 h for the detection and identification of low numbers of bacteria.2+ fluorescence PCR-SSCP seems to be a promising method for the differentiation of a broad range of pathogens found in usually sterile clinical sites, such as blood and cerebrospinal fluid.
Fusobacterium nucleatum is classified into five subspecies that inhabit the human oral cavity (F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, F. nucleatum subsp. fusiforme, F. nucleatum subsp. vincentii, and F. nucleatum subsp. animalis) based on several phenotypic characteristics and DNA-DNA hybridization patterns. However, the methods for detecting or discriminating the clinical isolates of F. nucleatum at the subspecies levels are laborious, expensive, and time-consuming. Therefore, in this study, the nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and zinc protease gene were analyzed to discriminate the subspecies of F. nucleatum. The partial sequences of rpoB (approximately 2,419 bp), the zinc protease gene (878 bp), and 16S rRNA genes (approximately 1,500 bp) of the type strains of five subspecies, 28 clinical isolates of F. nucleatum, and 10 strains of F. periodonticum (as a control group) were determined and analyzed. The phylogenetic data showed that the rpoB and zinc protease gene sequences clearly delineated the subspecies of F. nucleatum and provided higher resolution than the 16S rRNA gene sequences in this respect. According to the phylogenetic analysis of rpoB and the zinc protease gene, F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme might be classified into a single subspecies. Five clinical isolates could be delineated as a new subspecies of F. nucleatum. The results suggest that rpoB and the zinc protease gene are efficient targets for the discrimination and taxonomic analysis of the subspecies of F. nucleatum.
PCR primers that target the bacterial 16S rRNA genes (or the tuf gene for the genus Enterococcus) were used to identify 10 putative bacterial pathogens in root canals with necrotic pulp. In addition, the associations of these microorganisms with symptoms and a history of diabetes mellitus were investigated. Microbial samples from the root canals of 24 teeth with necrotic pulp were included in the study. PCR with universal bacterial primers identified bacterial DNA in 22 specimens; the remaining 2 specimens were from intact teeth that had been traumatized 6 months prior to treatment. PCR with specific primers showed that preoperative symptoms were significantly associated with the presence of Streptococcus spp. (P < 0.001 by chi-square analysis). There was also a nonsignificant trend for symptoms to be associated with Fusobacterium nucleatum and Porphyromonas gingivalis (odds ratio, >2) and for diabetes mellitus to be associated with P. gingivalis and Porphyromonas endodontalis (odds ratio, >2). Cloning and sequencing of the universal PCR product in one specimen revealed the presence of an organism related to the genus Olsenella, which has not previously been described in endodontic infections.
Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. pluton by sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae.
Fusobacterium nucleatum subsp. nucleatum has been associated with a variety of oral and nonoral infections such as periodontitis, pericarditis, bone infections, and brain abscesses. Several studies have shown the role of plasmin, a plasma serine protease, in increasing the invasive capacity of microorganisms. In this study, we investigated the binding of human plasminogen to F. nucleatum subsp. nucleatum, and its subsequent activation into plasmin. Plasminogen-binding activity of bacterial cells was demonstrated by a solid-phase dot blot assay using an anti-plasminogen antibody. The binding activity was heat resistant and involved cell-surface lysine residues since it was abolished in the presence of the lysine analog ɛ-aminocaproic acid. Activation of plasminogen-coated bacteria occurred following incubation with either streptokinase, urokinase-type plasminogen activator (u-PA), or a Porphyromonas gingivalis culture supernatant. In the case of the P. gingivalis culture supernatant, a cysteine protease was likely involved in the activation. The plasmin activity generated on the cell surface of F. nucleatum subsp. nucleatum could be inhibited by aprotinin. Activation of plasminogen by u-PA was greatly enhanced when plasminogen was bound to bacteria rather than in a free soluble form. u-PA-activated plasminogen-coated F. nucleatum subsp. nucleatum was found to degrade fibronectin, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tissue inhibitor of metalloproteinase-1 was also degraded by the plasmin activity generated on the bacterial cells. This study suggests a possible role for plasminogen, which is present in affected periodontal sites, in promoting tissue destruction and invasion by nonproteolytic bacteria such as F. nucleatum subsp. nucleatum.
A new molecular biological approach for the identification of bacteria is described. This approach employs PCR of bacterial cell lysates with conserved primers located in the 16S rRNA sequence flanking a variable region, and analysis of the amplified product was based on the principle of single-strand conformation polymorphism (SSCP). The PCR product was denatured and separated on a nondenaturing polyacrylamide gel. SSCP patterns were detected by silver staining the nucleic acids. The mobility of the single-stranded DNA is sequence dependent and could be used to identify the unknown bacteria. Feasibility of the technique was demonstrated for a broad panel of gram-negative and gram-positive bacteria. We tested over 100 strains of bacteria representing 15 genera and 40 species. With the use of only two primer sets, P11P-P13P and ER10-ER11, we were capable to discriminate the tested species at the genus and species levels. Species-specific patterns were obtained for, e.g., Clostridium spp., Listeria spp., Pseudomonas spp., and Enterobacter spp. PCR-SSCP is a sensitive technique; e.g., the sensitivity obtained for Escherichia coli cells was 30 CFU. This technique is a simple and rapid method for the detection and identification of a wide spectrum of bacteria by whole-cell-based PCR amplification with the use of conserved primers and identification by nondenaturing gel electrophoresis.
The bacterial microfloras of 8 healing and 10 nonhealing chronic venous leg ulcers were compared by using a combination of cultural analysis and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene products. Cultural analysis of the microflora revealed that the majority of both wound types carried the aerobes Staphylococcus and Pseudomonas spp. (89 and 80%, respectively). Sequencing of 16S ribosomal DNAs selected on the basis of DGGE profiling allowed the identification of strains not detected by cultural means. Of considerable interest was the finding that more than 40% of the sequences represented organisms not cultured from the wound from which they were amplified. DGGE profiles also revealed that all of the wounds possessed one apparently common band, identified by sequencing as Pseudomonas sp. The intensity of this PCR signal suggested that the bacterial load of nonhealing wounds was much higher for pseudomonads compared to healing wounds and that it may have been significantly underestimated by cultural analysis. Hence, the present study shows that DGGE could give valuable additional information about chronic wound microflora that is not apparent from cultural analysis alone.
Intracystic fluid was aseptically collected from 11 patients with postoperative maxillary cyst (POMC), and DNA was extracted from the POMC fluid. Bacterial species were identified by sequencing after cloning of approximately 580 bp of the 16S rRNA gene. Identification of pathogenic bacteria was also performed by culture methods. The phylogenetic identity was determined by sequencing 517–596 bp in each of the 1139 16S rRNA gene clones. A total of 1114 clones were classified while the remaining 25 clones were unclassified. A total of 103 bacterial species belonging to 42 genera were identified in POMC fluid samples by 16S rRNA gene analysis. Species of Prevotella (91%), Neisseria (73%), Fusobacterium (73%), Porphyromonas (73%), and Propionibacterium (73%) were found to be highly prevalent in all patients. Streptococcus mitis (64%), Fusobacterium nucleatum (55%), Propionibacterium acnes (55%), Staphylococcus capitis (55%), and Streptococcus salivarius (55%) were detected in more than 6 of the 11 patients. The results obtained by the culture method were different from those obtained by 16S rRNA gene analysis, but both approaches may be necessary for the identification of pathogens, especially of bacteria that are difficult to detect by culture methods, and the development of rational treatments for patients with POMC.
Segments of the ospA gene of Borrelia burgdorferi were amplified by the polymerase chain reaction (PCR). Oligonucleotide primers used in the reaction flank a 309-base-pair segment within the ospA gene. After 35 cycles of amplification, the product could be detected by agarose gel electrophoresis or dot hybridization with a 32P-labeled probe. This segment was amplified in all strains of B. burgdorferi tested, but it was not detected in other bacterial species. An additional primer pair which has a broad specificity for conserved 16S rRNA sequences that are present in eubacteria amplified a 215-base-pair fragment in all organisms tested. The sensitivity of PCR for the detection of B. burgdorferi in clinical samples was evaluated by seeding blood and urine specimens with B. burgdorferi and subjecting them to amplification. We were able to detect 10 organisms per ml of blood or urine, using PCR with dot hybridization detection. DNA extraction is not required for sample preparation. Blood and urine specimens were obtained from canines with clinical and serologic evidence of Lyme disease and subjected to PCR analysis. Of 17 clinical specimens from 15 animals, one blood specimen showed reactivity in the PCR.
We developed a real-time-PCR assay utilizing a molecular-beacon probe for the detection of Entamoeba histolytica and compared its sensitivity to stool antigen detection and traditional PCR. A total of 205 stool and liver abscess pus specimens from patients and controls were used for this purpose, 101 (49%) of which were positive by the TechLab E. histolytica-specific antigen detection test, while the other 104 (51%) stool and liver abscess pus specimens were negative by the antigen detection test. DNA was extracted from the stool and liver abscess pus specimens by the QIAGEN method and the small-subunit rRNA gene of E. histolytica and then amplified by traditional and real-time PCR. Out of these 205 stool and liver abscess pus specimens, 124 were positive by the real-time-PCR assay and 90 were positive by the traditional-PCR test. Compared to the real-time-PCR assay, the antigen detection test was 79% sensitive and 96% specific. When the traditional-PCR test results were compared to the real-time-PCR assay, the sensitivity of traditional PCR was 72% and the specificity was 99%. In conclusion, all three methods for the detection of E. histolytica were highly specific, with real-time PCR being the most sensitive.
The bacteria found in carious dentine were correlated with the tissue response of the dental pulps of 65 teeth extracted from patients with advanced caries and pulpitis. Standardized homogenates of carious dentine were plated onto selective and nonselective media under anaerobic and microaerophilic conditions. In addition, real-time PCR was used to quantify the recovery of anaerobic bacteria. Primers and fluorogenic probes were designed to detect the total anaerobic microbial load, the genera Prevotella and Fusobacterium, and the species Prevotella melaninogenica, Porphyromonas endodontalis, Porphyromonas gingivalis, and Micromonas (formerly Peptostreptococcus) micros. The pulpal pathology was categorized according to the cellular response and degenerative changes. Analysis of cultured bacteria showed a predominance of gram-positive microorganisms, particularly lactobacilli. Gram-negative bacteria were also present in significant numbers with Prevotella spp., the most numerous anaerobic group cultured. Real-time PCR analysis indicated a greater microbial load than that determined by colony counting. The total number of anaerobes detected was 41-fold greater by real-time PCR than by colony counting, while the numbers of Prevotella and Fusobacterium spp. detected were 82- and 2.4-fold greater by real-time PCR than by colony counting, respectively. Real-time PCR also identified M. micros, P. endodontalis, and P. gingivalis in 71, 60, and 52% of carious samples, respectively. Correlation matrices of the real-time PCR data revealed significant positive associations between M. micros and P. endodontalis detection and inflammatory degeneration of pulpal tissues. These anaerobes have been strongly implicated in endodontic infections that occur as sequelae to carious pulpitis. Accordingly, the data suggest that the presence of high levels of these bacteria in carious lesions may be indicative of irreversible pulpal pathology.
We have evaluated the use of a broad-range PCR aimed at the 16S rRNA gene in detecting bacterial meningitis in a clinical setting. To achieve a uniform DNA extraction procedure for both gram-positive and gram-negative organisms, a combination of physical disruption (bead beating) and a silica-guanidiniumthiocyanate procedure was used for nucleic acid preparation. To diminish the risk of contamination as much as possible, we chose to amplify almost the entire 16S rRNA gene. The analytical sensitivity of the assay was approximately 1 × 102 to 2 × 102 CFU/ml of cerebrospinal fluid (CSF) for both gram-negative and gram-positive bacteria. In a prospective study of 227 CSF samples, broad-range PCR proved to be superior to conventional methods in detecting bacterial meningitis when antimicrobial therapy had already started. Overall, our assay showed a sensitivity of 86%, a specificity of 97%, a positive predictive value of 80%, and a negative predictive value of 98% compared to culture. We are currently adapting the standard procedures in our laboratory for detecting bacterial meningitis; broad-range 16S ribosomal DNA PCR detection is indicated when antimicrobial therapy has already started at time of lumbar puncture or when cultures remain negative, although the suspicion of bacterial meningitis remains.
Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients.
We designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broad-range fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach.
PCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background.
While PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples.
Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5′ end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR–single-stranded DNA approach for microbial community analysis.
A novel approach was developed to quantify rRNA sequences in complex bacterial communities. The main bacterial 16S rRNAs in Drentse A grassland soils (The Netherlands) were amplified by reverse transcription (RT)-PCR with bacterium-specific primers and were separated by temperature gradient gel electrophoresis (TGGE). The primer pair used (primers U968-GC and L1401) was found to amplify with the same efficiency 16S rRNAs from bacterial cultures containing different taxa and cloned 16S ribosomal DNA amplicons from uncultured soil bacteria. The sequence-specific efficiency of amplification was determined by monitoring the amplification kinetics by kinetic PCR. The primer-specific amplification efficiency was assessed by competitive PCR and RT-PCR, and identical input amounts of different 16S rRNAs resulted in identical amplicon yields. The sequence-specific detection system used for competitive amplifications was TGGE, which also has been found to be suitable for simultaneous quantification of more than one sequence. We demonstrate that this approach can be applied to TGGE fingerprints of soil bacteria to estimate the ratios of the bacterial 16S rRNAs.
The microflora associated with three dentoalveolar abscesses was determined by cultural and molecular methods. 16S rRNA genes were randomly amplified by means of conserved eubacterial primers and cloned. Restriction fragment length polymorphism analysis of the clones and amplified genes encoding 16S rRNA from the cultured bacteria was used to detect putative unculturable bacteria. Clones representative of five predominant groups of uncultured organisms were sequenced. Two were identified as Porphyromonas gingivalis and Prevotella oris, and one was found to be closely related to Peptostreptococcus micros. The remaining two clones did not correspond to known, previously sequenced organisms. One was related to Zoogloea ramigera, a species of aerobic waterborne organisms, while the other was distantly related to the genus Prevotella. This study has demonstrated the possibility of the characterization of microflora associated with human infection by molecular methods without the inherent biases of culture.
Previous studies investigating microbial diversity in the Octopus Spring cyanobacterial mat community (Yellowstone National Park) have shown a discrepancy between bacterial populations observed by molecular retrieval and cultivation techniques. To investigate how selective enrichment culture techniques affect species composition, we used denaturing gradient gel electrophoresis (DGGE) separation of PCR-amplified 16S rRNA gene fragments to monitor the populations contained within enrichment cultures of aerobic chemoorganotrophic bacteria from the ca. 50 degrees C region of the mat community. By varying the degree of dilution of the inoculum, medium composition, and enrichment conditions and duration and by analyzing the cultures by DGGE, we detected 14 unique 16S rRNA sequence types. These corresponded to alpha-, beta-, gamma-, and delta-proteobacteria, Thermus relatives, and gram-positive bacteria with high G + C ratio and, at the highest inoculum dilutions, Chloroflexus aurantiacus relatives, which were estimated to still be approximately 300 times less abundant than cells of the mat primary producer, Synechococcus spp. Only three of these populations were previously cultivated on solidified medium after similar enrichment. Only two of these population have 16S rRNA sequences which were previously cloned directly from the mat. These results reveal a diversity of bacterial populations in enrichment culture which were not detected by either molecular retrieval or strain purification techniques.
These studies determined the characteristics of tissue destruction in a murine abscess model elicited by mixed infection with the periodontopathogens Fusobacterium nucleatum and Porphyromonas gingivalis. The interbacterial effects of this synergism, the kinetics of the relationship of the bacterial interaction, and the characteristics of the bacteria required for the tissue destruction were studied. Infection of mice with P. gingivalis and F. nucleatum strains elicited lesions of various sizes as a function of infective dose. Primary infection with F. nucleatum plus P. gingivalis at various ratios (i.e., <1:1) resulted in a significantly greater lesion size (P < 0.001) compared with that resulting from primary infection with P. gingivalis alone. At F. nucleatum/P. gingivalis ratios of > or = 1:1, spreading lesion formation and progression were significantly (P < 0.001) decreased, suggesting that bacterial interaction (i.e., coaggregation) may have inhibited the spread of the P. gingivalis infection to a site distant from the initial injection. Infection with F. nucleatum and P. gingivalis simultaneously (at different sites) or F. nucleatum administered within 4 h prior to or 1 h following P. gingivalis infection significantly enhanced the ability of P. gingivalis to form large phlegmonous lesions. Chemical inhibition of the P. gingivalis trypsin-like protease activity or the use of a trypsin-negative P. gingivalis strain abrogated tissue destruction either alone or in combination with F. nucleatum. Therefore, it was possible to examine aspects of virulence of these pathogens in a murine lesion model by either altering bacterial ratios, manipulating the time of infection, or targeting vital bacterial virulence factors.
The aim of the present study was to develop a monoclonal antibody that recognizes the shared antigen of Porphyromonas endodontalis so that we could use the antibody in direct identification and detection of P. endodontalis in infectious material from apical periodontal patients. We established a hybridoma cell line producing monoclonal antibody (BEB5) specific for P. endodontalis. BEB5 antibody reacted with all of the P. endodontalis strains tested, but not with any of the other black-pigmented Porphyromonas and Bacteroides spp. The antibody reacted specifically with the lipopolysaccharide (LPS) of three P. endodontalis strains of different serotypes (O1K1, O1K2, and O1K-). Western blotting (immunoblotting) analysis confirmed the specificity of the antibody to these LPSs, because the antibody recognized the typical "repetitive ladder" pattern characteristic of LPS on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. These observations demonstrate that P. endodontalis LPS is the shared antigen of this species. The antibody can specifically identify P. endodontalis on nitrocellulose membrane blots of bacterial colonies grown on agar. The antibody is also capable of directly detecting the presence of P. endodontalis in infectious material by immunoslot blot assay. These results indicate that LPS is the shared antigen of P. endodontalis and that BEB5 antibody against LPS is a useful one for direct identification and detection of the organisms in samples from apical periodontal patients.
Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database.
The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.
Broad-range real-time PCR and sequencing of the 16S rRNA gene region is a widely known method for the detection and identification of bacteria in clinical samples. However, because of the need for sequencing, such identification of bacteria is time-consuming. The aim of our study was to develop a more rapid 16S real-time PCR-based identification assay using species- or genus-specific probes. The Gram-negative bacteria were divided into Pseudomonas species, Pseudomonas aeruginosa, Escherichia coli, and other Gram-negative species. Within the Gram-positive species, probes were designed for Staphylococcus species, Staphylococcus aureus, Enterococcus species, Streptococcus species, and Streptococcus pneumoniae. The assay also included a universal probe within the 16S rRNA gene region for the detection of all bacterial DNA. The assay was evaluated with a collection of 248 blood cultures. In this study, the universal probe and the probes targeting Pseudomonas spp., P. aeruginosa, E. coli, Streptococcus spp., S. pneumoniae, Enterococcus spp., and Staphylococcus spp. all had a sensitivity and specificity of 100%. The probe specific for S. aureus showed eight discrepancies, resulting in a sensitivity of 100% and a specificity of 93%. These data showed high agreement between conventional testing and our novel real-time PCR assay. Furthermore, this assay significantly reduced the time needed for identification. In conclusion, using pathogen-specific probes offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.