| 1. |
- Fachmann, M S R, et al.
(författare)
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Detection of Salmonella in meat in less than 5 hours by a low-cost and non-complex sample preparation method.
- 2017
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 83:5
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Tidskriftsartikel (refereegranskat)abstract
- Salmonella is recognised as one of the most important foodborne bacteria, and has a wide health and socioeconomical impact worldwide. Fresh pork meat is one of the main sources of Salmonella and efficient and fast methods for detection are therefore necessary. Current methods for Salmonella detection in fresh meat usually include >16 h of culture enrichment, in few cases < 12 h, thus requiring at least two working shifts. Here, we report a rapid (< 5 h) and high throughput method, for screening of Salmonella in samples from fresh pork meat, consisting of a 3-h enrichment in standard buffered peptone water, and a real-time PCR compatible sample preparation method, based on filtration, centrifugation, and enzymatic digestion, followed by fast cycling real-time PCR detection. The method was validated in an un-paired, comparative study against the Nordic Committee on Food Analysis (NMKL) reference culture method 187. Pork meat samples (n=140) were either artificially contaminated with Salmonella at levels: 0, 1-10 and 10-100 CFU/25 g, or naturally contaminated. Cohen's Kappa for degree of agreement between the rapid method and the reference was 0.64 and the relative accuracy, sensitivity and specificity for the rapid method were 81.4, 95.1 and 97.9 %, respectively. The limit of detection (LOD50) was 8.8 CFU/25 g for the rapid method and 7.7 CFU/25 g for the reference method. Implementation of this method will enable faster release of Salmonella low risk meat, providing savings for meat producers, and help contribute to improved food safety.IMPORTANCE: While the cost of analysis and hands-on time of the presented rapid method were comparable to reference culture methods, the fast product release by this method can provide the meat industry with a competitive advantage. Not only will the abattoirs save costs for work hours and cold storage; consumers as well as retailers will also benefit from fresher meat with a longer shelf life. Furthermore, the presented sample preparation might be adjusted for application in detection of other pathogenic bacteria in different sample types.
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| 2. |
- Becker, K., et al.
(författare)
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Antibacterial activity of apramycin at acidic pH warrants wide therapeutic window in the treatment of complicated urinary tract infections and acute pyelonephritis
- 2021
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Ingår i: EBioMedicine. - : Elsevier B.V.. - 2352-3964. ; 73
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Tidskriftsartikel (refereegranskat)abstract
- Background: The clinical-stage drug candidate EBL-1003 (apramycin) represents a distinct new subclass of aminoglycoside antibiotics for the treatment of drug-resistant infections. It has demonstrated best-in-class coverage of resistant isolates, and preclinical efficacy in lung infection models. However, preclinical evidence for its utility in other disease indications has yet to be provided. Here we studied the therapeutic potential of EBL-1003 in the treatment of complicated urinary tract infection and acute pyelonephritis (cUTI/AP). Methods: A combination of data-base mining, antimicrobial susceptibility testing, time-kill experiments, and four murine infection models was used in a comprehensive assessment of the microbiological coverage and efficacy of EBL-1003 against Gram-negative uropathogens. The pharmacokinetics and renal toxicology of EBL-1003 in rats was studied to assess the therapeutic window of EBL-1003 in the treatment of cUTI/AP. Findings: EBL-1003 demonstrated broad-spectrum activity and rapid multi-log CFU reduction against a phenotypic variety of bacterial uropathogens including aminoglycoside-resistant clinical isolates. The basicity of amines in the apramycin molecule suggested a higher increase in positive charge at urinary pH when compared to gentamicin or amikacin, resulting in sustained drug uptake and bactericidal activity, and consequently in potent efficacy in mouse infection models. Renal pharmacokinetics, biomarkers for toxicity, and kidney histopathology in adult rats all indicated a significantly lower nephrotoxicity of EBL-1003 than of gentamicin. Interpretation: This study provides preclinical proof-of-concept for the efficacy of EBL-1003 in cUTI/AP. Similar efficacy but lower nephrotoxicity of EBL-1003 in comparison to gentamicin may thus translate into a higher safety margin and a wider therapeutic window in the treatment of cUTI/API. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section. © 2021 The Author(s)
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| 3. |
- Löfström, Charlotta, et al.
(författare)
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Fluorescence-based real-time quantitative polymerase chain reaction (qPCR) technologies for high throughput screening of pathogens
- 2014
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Ingår i: High Throughput Screening for Food Safety Assessment: Biosensor Technologies, Hyperspectral Imaging and Practical Applications. - : Elsevier Inc.. - 9780857098078 - 9780857098016 ; , s. 219-248
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Real-time quantitative polymerase chain reaction (qPCR) technology is being widely used for high throughput diagnostics of pathogens in the food production chain as an alternative to the more time-consuming and laborious culture-based standard methods. This chapter discusses the basics of qPCR, including data analysis, PCR instruments, and detection chemistries. The importance of choosing appropriate pre-PCR processing strategies - i.e., sampling, sample preparation, and PCR chemistry - to avoid PCR inhibition and achieve correct quantification is furthermore included. Major novel trends such as portable PCR instruments for in-field diagnostics, high-resolution melting curve analysis, and digital PCR are addressed. Finally, some perspectives on future trends are given. © 2015 Elsevier Ltd. All rights reserved.
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| 4. |
- Ågren, Joakim, et al.
(författare)
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In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences
- 2013
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Ingår i: Virulence. - : Taylor and Francis Inc.. - 2150-5594 .- 2150-5608. ; 4:8
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Tidskriftsartikel (refereegranskat)abstract
- Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays. © 2013 Landes Bioscience.
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| 5. |
- Josefsen, M. H., et al.
(författare)
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Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time pcr and propidium monoazide treatment, as a tool for quantitative risk assessment
- 2010
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 76:15, s. 5097-5104
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Tidskriftsartikel (refereegranskat)abstract
- A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method's performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (CT.) values (R2 = 0.993), with a quantification range of 1 × 102 to 1 × 107 CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R 2 = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment. Copyright © 2010, American Society tor Microbiology. All Rights Reserved.
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