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Journal of Clinical Microbiology Mar 1996The microflora associated with three dentoalveolar abscesses was determined by cultural and molecular methods. 16S rRNA genes were randomly amplified by means of...
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.
Topics: Adult; Bacteria; Base Sequence; Humans; Male; Molecular Sequence Data; Periapical Abscess; Polymorphism, Restriction Fragment Length; RNA, Ribosomal, 16S
PubMed: 8904410
DOI: 10.1128/jcm.34.3.537-542.1996 -
Revista Espanola de Quimioterapia :... Aug 2023
Topics: Humans; Prevotella; Lung Diseases; Bacteroidaceae Infections
PubMed: 37184104
DOI: 10.37201/req/001.2023 -
Journal of Thoracic Disease Oct 2019Culture-independent methods such as quantitative polymerase chain reaction (qPCR) are more sensitive for detecting pathogens than conventional culture. This study aimed...
Potential clinical utility of multiple target quantitative polymerase chain reaction (qPCR) array to detect microbial pathogens in patients with chronic obstructive pulmonary disease (COPD).
BACKGROUND
Culture-independent methods such as quantitative polymerase chain reaction (qPCR) are more sensitive for detecting pathogens than conventional culture. This study aimed to test the clinical potential of a multiple target qPCR array in identifying sputum pathogens, compared to traditional culture.
METHODS
Forty chronic obstructive pulmonary disease (COPD) patients provided spontaneous sputum and blood samples during an exacerbation event (n=25 patients) and in stable state (n=15 patients). Sputum was processed and analysed by microscopy, culture and sensitivity testing (MCS) to identify living microbial isolates, and multiple target qPCR (44 targets for bacterial and fungal pathogens and antibiotic resistance genes), and 16S rRNA gene sequencing.
RESULTS
Six microbial isolates (5 bacterial, 1 fungal) were cultured from 20 exacerbation and 10 stable patient sputum samples. Four of these microbial isolates had their presence in patient sputum confirmed by qPCR. All bacterial targets detected by qPCR were further confirmed by 16S rRNA gene sequencing at a genus level. qPCR identified significantly more bacterial pathogens than culture (P<0.001). The most prevalent bacterial species identified by qPCR were (72% of patients), (40%), (32%) and (17%). Microbial species diversity and richness were not significantly different between samples obtained from exacerbating and clinically stable cases. 16S rRNA gene sequencing identified (P=0.022, FDR =0.043 AUC =0.72) as a significantly different bacterial OTU (operational taxonomic units) in exacerbation sputum samples compared to stable state samples.
CONCLUSIONS
Multiple target qPCR was more sensitive for detection of sputum pathogens in COPD patients than conventional culture. 16S rRNA gene sequencing confirmed the identity at a genus level of all bacterial targets detected by qPCR, as well as identifying bacterial OTUs that could potentially be used to distinguish between exacerbation and stable COPD disease states. Multiple target qPCR pathogen detection in the sputum of COPD patients warrants further investigation to determine how it may influence COPD clinical management.
PubMed: 31737352
DOI: 10.21037/jtd.2019.10.39 -
BMC Infectious Diseases Sep 2021Descending necrotizing mediastinitis (DNM) is one of the most virulent forms of mediastinitis. The main causes of high mortality in DNM are believed to stem from...
BACKGROUND
Descending necrotizing mediastinitis (DNM) is one of the most virulent forms of mediastinitis. The main causes of high mortality in DNM are believed to stem from difficulty and delay in the diagnosis. Fast and accurate identification of pathogens is important for the treatment of these patients. Metagenomics next-generation sequencing (mNGS) is a powerful tool to identify all kinds of pathogens, especially for rare and complex infections.
CASE PRESENTATION
A 64-year-old male patient was admitted to the intensive care unit (ICU) with unconsciousness, dyspnea, and swelling in the mandible and neck. Computed tomography (CT) scan results combined with clinical laboratory examination indicated DNM. Vancomycin and imipenem were used, and vacuum sealing drainage was applied for debridement and drainage of the infected area. The positive mNGS results of drainage fluid confirmed the presence of mixed infection caused by Streptococcus anginosus, Prevotella oris, and several other anaerobes. The antibiotics were adjusted to piperacillin/tazobactam and tinidazole according to the mNGS results and antimicrobial susceptibility testing of cultured pathogens. After 11 days of antibiotic therapy, the infection symptoms of the neck and mediastinum improved, and the patient was transferred out of the ICU on the 26 day after negative result of drainage fluid culture.
CONCLUSION
This case suggested that mNGS is a promising technology for precise and fast pathogens identification with high sensitivity, which may guide the diagnosis of infectious diseases in the future trend.
Topics: Coinfection; High-Throughput Nucleotide Sequencing; Humans; Male; Mediastinitis; Metagenomics; Middle Aged; Necrosis; Prevotella
PubMed: 34479479
DOI: 10.1186/s12879-021-06624-4 -
Journal of Clinical Microbiology Sep 2007Multiple-displacement amplification (MDA) has been used to uniformly amplify bacterial genomes present in small samples, providing abundant targets for molecular...
Multiple-displacement amplification (MDA) has been used to uniformly amplify bacterial genomes present in small samples, providing abundant targets for molecular analysis. The purpose of this investigation was to combine MDA and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections. Sixty-six samples were collected from teeth with endodontic infections. Nonamplified and amplified samples were analyzed by checkerboard DNA-DNA hybridization for levels and proportions of 77 bacterial taxa. Counts, percentages of DNA probe counts, and percentages of teeth colonized for each species in amplified and nonamplified samples were computed. Significance of differences for each species between amplified and nonamplified samples was sought with Wilcoxon signed-rank test and adjusted for multiple comparisons. The amount of DNA in the samples ranged from 6.80 (+/- 5.2) ng before to 6.26 (+/- 1.73) mug after MDA. Seventy of the 77 DNA probes hybridized with one or more of the nonamplified samples. All probes hybridized with at least one sample after amplification. Most commonly detected species at levels of >10(4) in both amplified and nonamplified samples were Prevotella tannerae and Acinetobacter baumannii at frequencies between 89 and 100% of samples. The mean number of species at counts of >10(4) in amplified samples was 51.2 +/- 2.2 and in nonamplified samples was 14.5 +/- 1.7. The endodontic microbiota was far more complex than previously shown, although microbial profiles at teeth with or without periradicular lesions did not differ significantly. Species commonly detected in endodontic samples included P. tannerae, Prevotella oris, and A. baumannii.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Bacteria; Bacterial Infections; Bacteriological Techniques; Biodiversity; Child; Colony Count, Microbial; Humans; Middle Aged; Nucleic Acid Amplification Techniques; Nucleic Acid Hybridization; Tooth Diseases
PubMed: 17634304
DOI: 10.1128/JCM.02618-06 -
PloS One 2011The complexity of the human microbiome makes it difficult to reveal organizational principles of the community and even more challenging to generate testable hypotheses....
The complexity of the human microbiome makes it difficult to reveal organizational principles of the community and even more challenging to generate testable hypotheses. It has been suggested that in the gut microbiome species such as Bacteroides thetaiotaomicron are keystone in maintaining the stability and functional adaptability of the microbial community. In this study, we investigate the interspecies associations in a complex microbial biofilm applying systems biology principles. Using correlation network analysis we identified bacterial modules that represent important microbial associations within the oral community. We used dental plaque as a model community because of its high diversity and the well known species-species interactions that are common in the oral biofilm. We analyzed samples from healthy individuals as well as from patients with periodontitis, a polymicrobial disease. Using results obtained by checkerboard hybridization on cultivable bacteria we identified modules that correlated well with microbial complexes previously described. Furthermore, we extended our analysis using the Human Oral Microbe Identification Microarray (HOMIM), which includes a large number of bacterial species, among them uncultivated organisms present in the mouth. Two distinct microbial communities appeared in healthy individuals while there was one major type in disease. Bacterial modules in all communities did not overlap, indicating that bacteria were able to effectively re-associate with new partners depending on the environmental conditions. We then identified hubs that could act as keystone species in the bacterial modules. Based on those results we then cultured a not-yet-cultivated microorganism, Tannerella sp. OT286 (clone BU063). After two rounds of enrichment by a selected helper (Prevotella oris OT311) we obtained colonies of Tannerella sp. OT286 growing on blood agar plates. This system-level approach would open the possibility of manipulating microbial communities in a targeted fashion as well as associating certain bacterial modules to clinical traits (e.g.: obesity, Crohn's disease, periodontal disease, etc).
Topics: Algorithms; Bacteroides; Bayes Theorem; Biofilms; DNA; Dental Plaque; Hot Temperature; Humans; Metagenome; Models, Biological; Models, Genetic; Models, Theoretical; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Periodontitis; Principal Component Analysis; Systems Biology
PubMed: 22163302
DOI: 10.1371/journal.pone.0028438 -
Cureus May 2020Empyema necessitasis (EN) also referred to as empyema necessitans (EN) is a rare complication of empyema that can involve soft tissues outside the pleural cavity and can...
Empyema necessitasis (EN) also referred to as empyema necessitans (EN) is a rare complication of empyema that can involve soft tissues outside the pleural cavity and can lead to brachial plexus injury. Although, brachial plexus injury most commonly occurs as a result of trauma, inflammation, or malignancies, but it has been rarely seen in EN. We are reporting a rare and challenging case of empyema necessitasis (EN) causing impingement of brachial plexus in a 42-year-old, type 2 diabetic patient, who initially presented to the hospital with left-sided pleuritic chest pain and acute onset of left upper limb weakness. Urgent CT brain ruled out an acute neurological insult. Chest radiograph and contrast-enhanced CT thorax revealed left-sided loculated effusion. MRI of the left upper limb showed impingement of brachial plexus by the inflammatory process of surrounding effusion. Ultrasound-guided aspiration of the encapsulated fluid was performed and culture analysis of the fluid was remarkable for Prevotella oris (sensitive strain) growth. After drainage of the infected fluid and with a prolonged course of antibiotics, the patient's neurological symptoms improved significantly. Hospital course was uncomplicated and further follow-up was unremarkable for any deterioration. Our objective is to emphasize on the prompt management with close follow-up in EN, which can present with life-threatening complications as seen in our case. Any delay can compromise the patient's health. As per our limited knowledge, this case has not been reported in the literature.
PubMed: 32596084
DOI: 10.7759/cureus.8267 -
Journal of Oral Microbiology 2015Usefulness of next-generation sequencing (NGS) in assessing bacteria associated with oral squamous cell carcinoma (OSCC) has been undermined by inability to classify...
BACKGROUND
Usefulness of next-generation sequencing (NGS) in assessing bacteria associated with oral squamous cell carcinoma (OSCC) has been undermined by inability to classify reads to the species level.
OBJECTIVE
The purpose of this study was to develop a robust algorithm for species-level classification of NGS reads from oral samples and to pilot test it for profiling bacteria within OSCC tissues.
METHODS
Bacterial 16S V1-V3 libraries were prepared from three OSCC DNA samples and sequenced using 454's FLX chemistry. High-quality, well-aligned, and non-chimeric reads ≥350 bp were classified using a novel, multi-stage algorithm that involves matching reads to reference sequences in revised versions of the Human Oral Microbiome Database (HOMD), HOMD extended (HOMDEXT), and Greengene Gold (GGG) at alignment coverage and percentage identity ≥98%, followed by assignment to species level based on top hit reference sequences. Priority was given to hits in HOMD, then HOMDEXT and finally GGG. Unmatched reads were subject to operational taxonomic unit analysis.
RESULTS
Nearly, 92.8% of the reads were matched to updated-HOMD 13.2, 1.83% to trusted-HOMDEXT, and 1.36% to modified-GGG. Of all matched reads, 99.6% were classified to species level. A total of 228 species-level taxa were identified, representing 11 phyla; the most abundant were Proteobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Actinobacteria. Thirty-five species-level taxa were detected in all samples. On average, Prevotella oris, Neisseria flava, Neisseria flavescens/subflava, Fusobacterium nucleatum ss polymorphum, Aggregatibacter segnis, Streptococcus mitis, and Fusobacterium periodontium were the most abundant. Bacteroides fragilis, a species rarely isolated from the oral cavity, was detected in two samples.
CONCLUSION
This multi-stage algorithm maximizes the fraction of reads classified to the species level while ensuring reliable classification by giving priority to the human, oral reference set. Applying the algorithm to OSCC samples revealed high diversity. In addition to oral taxa, a number of human, non-oral taxa were also identified, some of which are rarely detected in the oral cavity.
PubMed: 26426306
DOI: 10.3402/jom.v7.28934 -
Infection and Immunity Nov 1992Activation of latent human fibroblast-type and neutrophil interstitial procollagenases as well as degradation of native type I collagen by supra- and subgingival dental...
Activation of latent human fibroblast-type and neutrophil interstitial procollagenases as well as degradation of native type I collagen by supra- and subgingival dental plaque extracts, an 80-kDa trypsinlike protease from Porphyromas gingivalis (ATCC 33277), a 95-kDa chymotrypsinlike protease from Treponema denticola (ATCC 29522), and selected bacterial species commonly isolated in periodontitis was studied. The bacteria included were Prevotella intermedia (ATCC 25261), Prevotella buccae (ES 57), Prevotella oris (ATCC 33573), Porphyromonas endodontalis (ES 54b), Actinobacillus actinomycetemcomitans (ATCC 295222), Fusobacterium nucleatum (ATCC 10953), Mitsuokella dentalis (DSM 3688), and Streptococcus mitis (ATCC 15909). None of the bacteria activated latent procollagenases; however, both sub- and supragingival dental plaque extracts (neutral salt extraction) and proteases isolated from cell extracts from potentially periodontopathogenic bacteria P. gingivalis and T. denticola were found to activate latent human fibroblast-type and neutrophil interstitial procollagenases. The fibroblast-type interstitial collagenase was more efficiently activated by bacterial proteases than the neutrophil counterpart, which instead preferred nonproteolytic activation by the oxidative agent hypochlorous acid. The proteases were not able to convert collagenase tissue inhibitor of metalloproteinase (TIMP-1) complexes into active form or to change the ability of TIMP-1 to inhibit interstitial collagenase. None of the studied bacteria, proteases from P. gingivalis and T. denticola, or extracts of supra- and subgingival dental plaque showed any significant collagenolytic activity. However, the proteases degraded native and denatured collagen fragments after cleavage by interstitial collagenase and gelatinase. Our results indicate that proteases from periodontopathogenic bacteria can act as direct proteolytic activators of human procollagenases and degrade collagen fragments. Thus, in concert with host enzymes the bacterial proteases may participate in periodontal tissue destruction.
Topics: Collagenases; Dental Plaque; Endopeptidases; Enzyme Activation; Fibroblasts; Glycoproteins; Humans; In Vitro Techniques; Matrix Metalloproteinase 1; Matrix Metalloproteinase 8; Neutrophils; Periodontitis; Tissue Inhibitor of Metalloproteinases; Treponema
PubMed: 1398963
DOI: 10.1128/iai.60.11.4491-4495.1992 -
BMC Microbiology May 2024We evaluated whether the sputum bacterial microbiome differs between nontuberculous mycobacteria pulmonary disease (NTM-PD) patients with stable disease not requiring... (Comparative Study)
Comparative Study
BACKGROUND
We evaluated whether the sputum bacterial microbiome differs between nontuberculous mycobacteria pulmonary disease (NTM-PD) patients with stable disease not requiring antibiotic treatment and those requiring antibiotics.
METHODS
We collected sputum samples from 21 clinically stable NTM-PD patients (stable group) and 14 NTM-PD patients needing antibiotic treatment (treatment group). We also obtained 13 follow-up samples from the stable group. We analyzed the 48 samples using 16S rRNA gene sequencing (V3-V4 region) and compared the groups.
RESULTS
In the linear discriminant analysis effect size (LEfSe) analysis, the species Porphyromonas pasteri, Haemophilus parahaemolyticus, Prevotella nanceiensis, and Gemella haemolysans were significantly more prevalent in the sputum of the stable group compared to the treatment group. No taxa showed significant differences in alpha-/beta-diversity or LEfSe between the 21 baseline and 13 follow-up sputum samples in the stable group. In the stable group, the genus Bergeyella and species Prevotella oris were less common in patients who achieved spontaneous culture conversion (n = 9) compared to those with persistent NTM positivity (n = 12) (effect size 3.04, p = 0.039 for Bergeyella; effect size 3.64, p = 0.033 for P. oris). In the treatment group, H. parainfluenzae was more common in patients with treatment success (n = 7) than in treatment-refractory patients (n = 7) (effect size 4.74, p = 0.013).
CONCLUSIONS
Our study identified distinct bacterial taxa in the sputum of NTM-PD patients based on disease status. These results suggest the presence of a microbial environment that helps maintain disease stability.
Topics: Humans; Sputum; Male; Female; Microbiota; Aged; Mycobacterium Infections, Nontuberculous; RNA, Ribosomal, 16S; Middle Aged; Anti-Bacterial Agents; Bacteria; Nontuberculous Mycobacteria; DNA, Bacterial; Lung Diseases
PubMed: 38760693
DOI: 10.1186/s12866-024-03308-2