| 1. |
- Dahlén, Gunnar, 1944, et al.
(författare)
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Methodological issues in the quantification of subgingival microorganisms using the checkerboard technique.
- 2015
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Ingår i: Journal of microbiological methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 110, s. 68-77
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Tidskriftsartikel (refereegranskat)abstract
- The reproducibility and reliability of quantitative microbiological assessments using the DNA-DNA hybridization "checkerboard method" (CKB) were assessed. The data originated from 180 chronic periodontitis patients, who were enrolled in a clinical trial and sampled at baseline, and 3 and 12m post-therapy. The samples were divided into two portions allowing evaluation of reproducibility. In total, 531 samples were analyzed in a first run, using standard bacterial preparations of cells and 513 samples were accessible for analysis in the second, using standards based on purified DNA from the species. The microbial probe panel consisted of periodontitis marker bacteria as well as non-oral microorganisms. Three different ways of quantifying and presenting data; the visual scoring method, VSM, the standard curve method, SCM, and the percent method, PM, were compared. The second set of analyses based on the use of standard preparations of pure DNA was shown to be more consistent than the first set using standards based on cells, while the effect of storage time per se up to 2.5y seemed to be marginal. The best reproducibility was found for Tannerella forsythia, irrespective of quantification technique (Spearman's rho=0.587, Pearson's r≥0.540). The percent method (PM) based on percent of High Standard (10(6) cells) was more reliable than SCM based on a linear calibration of the High Standard and a Low Standard (10(5) cells). It was concluded that the reproducibility of the CBK method varied between different bacteria. High quality and pure specific DNA whole genomic probes and standards may have a stronger impact on the precision of the data than storage time and conditions.
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| 2. |
- Fridlund, Jimmy, et al.
(författare)
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A microbiological method for determining serum levels of broad spectrum β-lactam antibiotics in critically ill patients
- 2016
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 129, s. 23-27
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Tidskriftsartikel (refereegranskat)abstract
- Background Recent studies show that suboptimal blood levels of β-lactam antibiotics are present in intensive care unit (ICU) patients. A common reference method for assessing drug concentrations is liquid chromatography coupled with mass-spectrometry (LC-MS) which is highly accurate but rarely available outside reference centres. Thus, our aim was to develop a microbiological method for monitoring β-lactam antibiotic serum levels which could be used at any hospital with a microbiological laboratory. Methods The method was developed as a 96-well broth microdilution format to assess the concentrations of cefotaxime (CTX), meropenem (MER), and piperacillin (PIP). Patient serum containing antibiotics were diluted in suspensions of bacteria with known minimal inhibitory concentrations (MICs). Serum antibiotic concentrations were calculated by dividing the MIC with the dilution factor at which the serum inhibited growth of the bacterial suspension. Serum (n = 88) from ICU patients at four hospitals in south-east Sweden were analysed and compared to LC-MS analysis. Results The overall accuracy and precision for spiked samples and patient samples was within the pre-set target of ± 20.0% for all drugs. There was a significant correlation between the microbiological assay and LC-MS for the patient samples (CTX: r = 0.86, n = 31; MER: r = 0.96, n = 11; PIP: r = 0.88, n = 39) and the agreement around the clinical cut-off for CTX (4.0 mg/l), MER (2.0 mg/l) and PIP (16.0 mg/l) was 90%, 100% and 87%, respectively. Conclusion The microbiological method has a performance for determination of serum levels of meropenem, piperacillin and cefotaxime suitable for clinical use. It is an inexpensive method applicable in any microbiology laboratory.
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| 3. |
- Håkansson, Sebastian
(författare)
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Survival and phosphate solubilisation activity of desiccated formulations of Penicillium bilaiae and Aspergillus niger influenced by water activity
- 2018
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 150, s. 39-46
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Tidskriftsartikel (refereegranskat)abstract
- The impact of formulation and desiccation on the shelf life of phosphate (P)-solubilising microorganisms is often under-studied, particularly relating to their ability to recover P-solubilisation activity. Here, Penicilllium bilaiae and Aspergillus niger were formulated on vermiculite (V) alone, or with the addition of protectants (skimmed milk (V + SM) and trehalose (V + T)), and on sewage sludge ash with (A + N) and without nutrients (A), and dried in a convective air dryer. After drying, the spore viability of P. bilaiae was greater than that of A. niger. V formulations achieved the highest survival rates without being improved by the addition of protectants. P. bilaiae formulated on V was selected for desiccation in a fluidised bed dryer, in which several temperatures and final water activities (aw) were tested. The highest spore viability was achieved when the formulation was dried at 25 degrees C to a final aw > 0.3. During three months' storage, convective air dried formulations were stable for both strains, except in the presence of skimmed milk for P. bilaiae which saw a decrease in spore viability. In the fluidised bed-dried formulations, when aw > 0.3, the loss in viability was higher, especially when stored at 20 degrees C, than at aw < 0.1. P-solubilisation activity performed on ash was preserved in most of the formulations after desiccation and storage. Overall, a low drying temperature and high final aw positively affected P. bilaiae viability, however a trade-off between higher viability after desiccation and shelf life should be considered. Further research is needed to optimise viability over time and on more sustainable carriers.
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| 4. |
- Ibrahim, Victor, et al.
(författare)
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Enzymatic monitoring of lignin and lignin derivatives biooxidation.
- 2016
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 120, s. 53-55
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Tidskriftsartikel (refereegranskat)abstract
- Lignin oxidation was enzymatically monitored by measuring methanol released during the reaction. The methanol was oxidized to formaldehyde and hydrogen peroxide, and the latter used to oxidize ABTS to a product measured spectrophotometrically. The efficiency was comparable to the commonly used gas chromatography method. The assay was fast and inexpensive.
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| 5. |
- Isaksson, Jenny, et al.
(författare)
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Comparison of multilocus sequence typing and multilocus typing microarray of Chlamydia trachomatis strains from Argentina and Chile
- 2016
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 127, s. 214-218
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Tidskriftsartikel (refereegranskat)abstract
- This study compared conventional ompA genotyping of Chlamydia trachomatis with multilocus sequence typing (MLST) and multilocus typing (MLT) DNA microarray. DNA extracts of 104 C trachomatis positive specimens were analyzed by ompA sequencing and MIST and of these 76 by MLT array. Obtained MIST sequence types (STs) were compared to sequences in the database http://mIstdb.uu.se. The resolution obtained for MIST (35 STs) was 2.1 higher than for ompA sequencing (17 variants) and 13 higher than MLT array (27 MLT groups). Among the 104 samples the predominant genotype E could be divided into 5 ompA variants and 23 STs of which 16 had not been reported in previous studies. The most common STs, ST3 and ST56, were identified as founders and are common in several countries on a global scale. The MIST and the MLT array provided similar strain discrimination capacity and showed considerably higher resolution than conventional ompA sequencing.
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| 6. |
- Koptina, Anna, et al.
(författare)
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Challenges to get axenic cultures of Trichomonas spp. : A new approach in eradication of contaminants and maintenance of laboratory microbiological cultures
- 2015
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 118, s. 25-30
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Tidskriftsartikel (refereegranskat)abstract
- Contamination of microbiological and cell cultures is a major problem in many scientific and clinical laboratories as well as bioproduct manufacturers worldwide. In the current study we established a rapid (9 day) method to detect and eliminate fungal and bacterial contamination in cultures of the unicellular eukaryote Trichomonas spp. The developed method combines identification of the contaminating microorganisms using PCR and sequencing of the 16/18S regions followed by phylogenetic analysis. The next step was a phylogeny-guided selection of antibiotic treatments. We then used a two-step propidium iodide-resorufin assay to test the effect of selected antibiotics. The result was a quick and worthwhile purification of trichomonad laboratory cultures. Our workflow may also be implemented to obtain new isolates of trichomonads from clinical samples if initial broad-spectrum antibiotic therapy fails.
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| 7. |
- Linder, Tomas
(författare)
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A standardized toolkit for genetic engineering of CTG Glade yeasts
- 2018
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 144, s. 152-156
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a series of synthetic constructs suitable to genetically manipulate a broad range of yeast species belonging to the fungal CTG Glade. This molecular toolbox notably allows heterologous gene expression, single or dual fluorescence labeling and construction of luciferase-expressing strains for bioluminescence imaging.
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| 8. |
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| 9. |
- Nestor, David, 1992-, et al.
(författare)
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Evaluation of the FilmArray (TM) Meningitis/Encephalitis panel with focus on bacteria and Cryptococcus spp
- 2019
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 157, s. 113-116
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Molecular methods provide fast and accurate detection of both bacteria and viruses in the cerebrospinal fluid (CSF) causing infection in the central nervous system (CNS). In the present study we evaluated the bacterial detection performance of the fully automated FilmArray (TM) Meningitis/Encephalitis (ME) panel (bioMerieux) by comparing it with culture and multiplexed in-house PCR. Methods: Three sample types were analysed; Contrived samples with known bacterial/fungal concentration (n = 29), clinical samples from patients with verified cause of CNS infection (n = 17) and external quality assessment (EQA) samples (n = 11). Another six samples were purposely prepared with multiple targets to evaluate multiplex capacity. Results: The FilmArray (TM) had a slightly higher limit of detection for Streptococcus pneumoniae, Neisseria meningitidis, Listeria monocytogenes and Streptococcus agalactiae compared to in-house PCR methods but performed equal or better when compared to culture. The FilmArray (TM) ME panel detected the expected pathogen in 17 of 17 clinical samples and yielded detection of three additional viruses of which one was confirmed with comparator techniques. All but one of the EQA samples were correctly detected. Conclusions: The results of this study are promising and the FilmArray (TM) ME panel could add to the diagnostic algorithm in CNS-infections. However, the limit of detection for the important pathogens N. meningitidis and S. pneumoniae could be improved.
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| 10. |
- Periyannan Rajeswari, Prem Kumar, et al.
(författare)
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Multiple pathogen biomarker detection using an encoded bead array in droplet PCR
- 2017
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Ingår i: Journal of Microbiological Methods. - : Elsevier. - 0167-7012 .- 1872-8359. ; 139, s. 22-28
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Tidskriftsartikel (refereegranskat)abstract
- We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color coded Luminex beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads.
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| 11. |
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| 12. |
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| 13. |
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| 14. |
- Sannö, Axel, et al.
(författare)
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The development of a screening protocol for Salmonella spp. and enteropathogenic Yersinia spp. in samples from wild boar (Sus scrofa) also generating MLVA-data for Y. enterocolitica and Y. pseudotuberculosis
- 2018
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 150, s. 32-38
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Tidskriftsartikel (refereegranskat)abstract
- Salmonellosis and yersiniosis are notifiable human diseases that are commonly associated with contaminated food. Domestic pigs as well as wild boars and other wild-life have been identified as reservoirs of these bacteria. Methods for cultivation and molecular epidemiological investigations of Salmonella spp. are well established, however, cultivation of enteropathogenic Yersinia spp. is time- consuming and the commonly used method for molecular epidemiological investigations, pulsed-field gel electrophoresis, lack in discriminatory power. The aim of this study was to develop and evaluate a screening protocol well suited for wildlife samples and other highly contaminated samples. The method is based on PCR-screening followed by Multiple Loci Variant number tandem repeat Analysis (MLVA) on enrichment broth to obtain molecular epidemiological data for enteropathogenic Yersinia spp. without the need for pure isolates. The performance of the protocol was evaluated using wild boar samples (n = 354) including tonsils, faeces and lymph nodes from 90 Swedish wild boars. The new protocol performed as well as or better than the established ISO-standards for detection and cultivation of Y. enterocolitica and Salmonella spp., however for cultivation of Y. pseudotuberculosis, further development is needed. The selection for motility seems beneficial for the enrichment of Salmonella spp. and Y. enterocolitica. Further, the selective enrichment prior to PCR-analysis eliminates inhibitory factors present in the original sample. In total, ten isolates of Y. enterocolitica of various bio-serotypes were obtained, and the MLVA-profile of these isolates were consistent with the profiles from the corresponding enrichment broth. Further, 22 isolates of Salmonella spp. comprising six different serovars were obtained with S. Fulica, S. Hadar and a monophasic S. Typhimurium being the most common. In conclusion, the presented screening protocol offers a rapid and efficient way to obtain prevalence data from a large sample set as well as MLVA-data within a short time frame. These results can hence improve the knowledge on the epidemiology and distribution of these pathogens and their importance to public health.
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| 15. |
- Ungphakorn, Wanchana, et al.
(författare)
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Evaluation of automated time-lapse microscopy for assessment of in vitro activity of antibiotics
- 2017
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 132, s. 69-75
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Tidskriftsartikel (refereegranskat)abstract
- This study aimed to evaluate the potential of a new time-lapse microscopy based method (oCelloScope) to efficiently assess the in vitro antibacterial effects of antibiotics. Two E. con and one P. aeruginosa strain were exposed to ciprofloxacin, colistin, ertapenem and meropenem in 24-h experiments. Background corrected absorption (BCA) derived from the oCelloScope was used to detect bacterial growth. The data obtained with the oCelloScope were compared with those of the automated Bioscreen C method and standard time-kill experiments and a good agreement in results was observed during 6-24 h of experiments. Viable counts obtained at 1, 4, 6 and 24 h during oCelloScope and Bioscreen C experiments were well correlated with the corresponding BCA and optical density (OD) data. Initial antibacterial effects during the first 6 h of experiments were difficult to detect with the automated methods due to their high detection limits (approximately 105 CFU/mL for oCelloScope and 107 CFU/mL for Bioscreen C), the inability to distinguish between live and dead bacteria and early morphological changes of bacteria during exposure to ciprofloxacin, ertapenem and meropenem. Regrowth was more frequently detected in time-kill experiments, possibly related to the larger working volume with an increased risk of preexisting or emerging resistance. In comparison with Bioscreen C, the oCelloScope provided additional information on bacterial growth dynamics in the range of 105 to 107 CFU/mL and morphological features. In conclusion, the oCelloScope would be suitable for detection of in vitro effects of antibiotics, especially when a large number of regimens need to be tested.
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