Optimization of quantitative polymerase chain reactions for detection and quantification of eight periodontal bacterial pathogens

BACKGROUND: The aim of this study was to optimize quantitative (real-time) polymerase chain reaction (qPCR) assays for 8 major periodontal pathogens, i.e. Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Parvimonas micros, Porphyromonas gingivalis, Prevotella intermedia, Tanerella forsythia and Treponema denticola, and of the caries pathogen Streptococcus mutans. RESULTS: Eighteen different primer pairs were analyzed in silico regarding specificity (using BLAST analysis) and the presence of secondary structures at primer binding sites (using mFOLD). The most specific and efficiently binding primer pairs, according to these analyses, were selected for qPCR-analysis to determine amplification efficiency, limit of quantification and intra-run reproducibility. For the selected primer pairs, one for each species, the specificity was confirmed by assessing amplification of DNA extracts from isolates of closely related species. For these primer pairs, the intercycler portability was evaluated on 3 different thermal cyclers (the Applied Biosystems 7300, the Bio-Rad iQ5 and the Roche Light Cycler 480). For all assays on the different cyclers, a good correlation of the standard series was obtained (i.e. r2 >= 0.98), but quantification limits varied among cyclers. The overall best quantification limit was obtained by using a 2 mul sample in a final volume of 10 mul on the Light Cycler 480. CONCLUSIONS: In conclusion, the proposed assays allow to quantify the bacterial loads of S. mutans, 6 periodontal pathogenic species and the genus Fusobacterium.This can be of use in assessing periodontal risk, determination of the optimal periodontal therapy and evaluation of this treatment

Effects of propidium monoazide (PMA) treatment on mycobiome and bacteriome analysis of cystic fibrosis airways during exacerbation

Introduction and Purpose : Propidium monoazide (PMA)-pretreatment has increasingly been applied to remove the bias from dead or damaged cell artefacts, which could impact the microbiota analysis by high-throughput sequencing. Our study aimed to determine whether a PMA-pretreatment coupled with high-throughput sequencing analysis provides a different picture of the airway mycobiome and bacteriome. Results and Discussion : We compared deep-sequencing data of mycobiota and microbiota of 15 sputum samples from 5 cystic fibrosis (CF) patients with and without prior PMA-treatment of the DNA-extracts. PMA-pretreatment had no significant effect on the entire and abundant bacterial community (genera expressed as operational taxonomic units (OTUs) with a relative abundance greater than or equal to 1%), but caused a significant difference in the intermediate community (less than 1%) when analyzing the alpha biodiversity Simpson index (p = 0.03). Regarding PMA impact on the airway mycobiota evaluated for the first time here; no significant differences in alpha diversity indexes between PMA-treated and untreated samples were observed. Regarding beta diversity analysis, the intermediate communities also differed more dramatically than the total and abundant ones when studying both mycobiome and bacteriome. Our results showed that only the intermediate (or low abundance) population diversity is impacted by PMA-treatment, and therefore that abundant taxa are mostly viable during acute exacerbation in CF. Given such a cumbersome protocol (PMA-pretreatment coupled with high-throughput sequencing), we discuss its potential interest within the follow-up of CF patients. Further studies using PMA-pretreatment are warranted to improve our "omic" knowledge of the CF airways

Influence of chronic azithromycin treatment on the composition of the oropharyngeal microbial community in patients with severe asthma

Background: This study of the oropharyngeal microbiome complements the previously published AZIthromycin in Severe ASThma (AZISAST) clinical trial, where the use of azithromycin was assessed in subjects with exacerbationprone severe asthma. Here, we determined the composition of the oropharyngeal microbial community by means of deep sequencing of the amplified 16S rRNA gene in oropharyngeal swabs from patients with exacerbationprone severe asthma, at baseline and during and after 6 months treatment with azithromycin or placebo. Results: A total of 1429 OTUs were observed, of which only 59 were represented by more than 0.02% of the reads. Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria and Actinobacteria were the most abundant phyla and Streptococcus and Prevotella were the most abundant genera in all the samples. Thirteen species only accounted for two thirds of the reads and two species only, i.e. Prevotella melaninogenica and Streptococcus mitis/pneumoniae, accounted for one fourth of the reads. We found that the overall composition of the oropharyngeal microbiome in patients with severe asthma is comparable to that of the healthy population, confirming the results of previous studies. Long term treatment (6 months) with azithromycin increased the species Streptococcus salivarius approximately 5-fold and decreased the species Leptotrichia wadei approximately 5-fold. This was confirmed by Boruta feature selection, which also indicated a significant decrease of L. buccalis/L. hofstadtii and of Fusobacterium nucleatum. Four of the 8 treated patients regained their initial microbial composition within one month after cessation of treatment. Conclusions: Despite large diversity of the oropharyngeal microbiome, only a few species predominate. We confirm the absence of significant differences between the oropharyngeal microbiomes of people with and without severe asthma. Possibly, long term azithromycin treatment may have long term effects on the composition of the oropharygeal microbiome in half of the patients

Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosa in sputum of cystic fibrosis patients

Background: Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in cystic fibrosis (CF) patients. Early aggressive antibiotic therapy has been shown to be effective in preventing chronic colonization. Therefore, early detection is important and sensitive detection methods are warranted. In this study, we used a dilution series of P. aeruginosa positive sputa, diluted in a pool of P. aeruginosa negative sputa, all from CF patients-to mimick as closely as possible the sputa sent to routine laboratories-to compare the sensitivity of three culture techniques versus that of two conventional PCR formats and four real-time PCR formats, each targeting the P. aeruginosa oprL gene. In addition, we compared five DNA-extraction protocols. Results: In our hands, all three culture methods and the bioMerieux easyMAG Nuclisens protocol Generic 2.0.1, preceded by proteinase K pretreatment and followed by any of the 3 real-time PCR formats with probes were most sensitive and able to detect P. aeruginosa up to 50 cfu/ml, i.e. the theoretical minimum of one cell per PCR mixture, when taking into account the volumes used in this study of sample for DNA-extraction, of DNA-elution and of DNA-extract in the PCR mixture. Conclusion: In this study, no difference in sensitivity could be found for the detection of P. aeruginosa from sputum between microbiological culture and optimized DNA-extraction and real-time PCR. The results also indicate the importance of the optimization of the DNA-extraction protocol and the PCR format

Comparison of culture and qPCR for the detection of Pseudomonas aeruginosa in not chronically infected cystic fibrosis patients

Pseudomonas aeruginosa is the major respiratory pathogen causing severe lung infections among CF patients, leading to high morbidity and mortality. Once infection is established, early antibiotic treatment is able to postpone the transition to chronic lung infection. In order to optimize the early detection, we compared the sensitivity of microbiological culture and quantitative PCR (qPCR) for the detection of P. aeruginosa in respiratory samples of not chronically infected CF patients.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Evaluation of the Engineering Company΄s Performance

Import 05/08/2014Cílem této diplomové práce je analyzovat a zhodnotit výkonnost podniku LUCCO a.s. pomocí vybraných metod a na základě zjištěných výsledků navrhnout doporučení pro případné zlepšení.V teoretické části jsou popsány pojmy výkonnost a jednotlivé metody tj. SWOT analýza, BSC, a bonitní a bankrotní modely, které se aplikují v praktické části. V praktické části diplomové práce se nachází charakteristika podniku LUCCO a.s. a následně zde budou zpracovány a popsány jednotlivé metody a okomentována výsledná zjištění. V poslední části jsou doporučení pro zlepšení výkonnosti podniku.The aim of this thesis is to analyze and evaluate the performance of the company as LUCCO using selected methods and based on the findings propose recommendations for possible zlepšení.V theoretical part describes the concepts and the performance of each method, ie, SWOT analysis, BSC, and value and bankruptcy models that are applied in the practical part. In the practical part of the thesis is characteristic of the enterprise as LUCCO and then there will be processed and described various methods and discussed the resulting findings. In the last section are recommendations for improving business performance.152 - Katedra podnikohospodářskávelmi dobř

The development of a 16S rRNA gene based PCR for the identification of Streptococcus pneumoniae and comparison with four other species specific PCR assays

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus pneumoniae </it>is one of the most frequently encountered pathogens in humans but its differentiation from closely related but less pathogenic streptococci remains a challenge.</p> <p>Methods</p> <p>This report describes a newly-developed PCR assay (Spne-PCR), amplifying a 217 bp product of the 16S rRNA gene of <it>S. pneumoniae</it>, and its performance compared to other genotypic and phenotypic tests.</p> <p>Results</p> <p>The new PCR assay designed in this study, proved to be specific at 57°C for <it>S. pneumoniae</it>, not amplifying <it>S. pseudopneumoniae </it>or any other streptococcal strain or any strains from other upper airway pathogenic species. PCR assays (psaA, LytA, ply, spn9802-PCR) were previously described for the specific amplification of <it>S. pneumoniae</it>, but <it>psaA</it>-PCR was the only one found not to cross-react with <it>S. pseudopneumoniae</it>.</p> <p>Conclusion</p> <p>Spne-PCR, developed for this study, and psaA-PCR were the only two assays which did not mis-identify <it>S. pseudopneumoniae </it>as <it>S. pneumoniae</it>. Four other PCR assays and the AccuProbe assay were unable to distinguish between these species.</p

Pseudomonas aeruginosa Population Structure Revisited

At present there are strong indications that Pseudomonas aeruginosa exhibits an epidemic population structure; clinical isolates are indistinguishable from environmental isolates, and they do not exhibit a specific (disease) habitat selection. However, some important issues, such as the worldwide emergence of highly transmissible P. aeruginosa clones among cystic fibrosis (CF) patients and the spread and persistence of multidrug resistant (MDR) strains in hospital wards with high antibiotic pressure, remain contentious. To further investigate the population structure of P. aeruginosa, eight parameters were analyzed and combined for 328 unrelated isolates, collected over the last 125 years from 69 localities in 30 countries on five continents, from diverse clinical (human and animal) and environmental habitats. The analysed parameters were: i) O serotype, ii) Fluorescent Amplified-Fragment Length Polymorphism (FALFP) pattern, nucleotide sequences of outer membrane protein genes, iii) oprI, iv) oprL, v) oprD, vi) pyoverdine receptor gene profile (fpvA type and fpvB prevalence), and prevalence of vii) exoenzyme genes exoS and exoU and viii) group I pilin glycosyltransferase gene tfpO. These traits were combined and analysed using biological data analysis software and visualized in the form of a minimum spanning tree (MST). We revealed a network of relationships between all analyzed parameters and non-congruence between experiments. At the same time we observed several conserved clones, characterized by an almost identical data set. These observations confirm the nonclonal epidemic population structure of P. aeruginosa, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise. One of these clones is the renown and widespread MDR serotype O12 clone. On the other hand, we found no evidence for a widespread CF transmissible clone. All but one of the 43 analysed CF strains belonged to a ubiquitous P. aeruginosa “core lineage” and typically exhibited the exoS+/exoU− genotype and group B oprL and oprD alleles. This is to our knowledge the first report of an MST analysis conducted on a polyphasic data set