| 1. |
- Chandler, Jeffrey C., et al.
(författare)
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Validation of a screening method for the detection of colistin-resistant E. coli containing mcr-1 in feral swine feces
- 2020
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Ingår i: Journal of Microbiological Methods. - : ELSEVIER. - 0167-7012 .- 1872-8359. ; 172
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Tidskriftsartikel (refereegranskat)abstract
- A method was developed and validated for the detection of colistin-resistant Escherichia coli containing mcr-1 in the feces of feral swine. Following optimization of an enrichment method using EC broth supplemented with colistin (1 mu g/mL) and vancomycin (8 mu g/mL), aliquots derived from 100 feral swine fecal samples were spiked with of one of five different mcr-1 positive E. coli strains (between 10(0) and 10(4) CFU/g), for a total of 1110 samples tested. Enrichments were then screened using a simple boil-prep and a previously developed real-time PCR assay for mcr-1 detection. The sensitivity of the method was determined in swine feces, with mcr-1 E. coli inocula of 0.1-9.99 CFU/g (n = 340), 10-49.99 CFU/g (n = 170), 50-99 CFU/g (n = 255), 100-149 CFU/g (n = 60), and 200-2200 CFU/g (n = 175), which were detected with 32%, 72%, 88%, 95%, and 98% accuracy, respectively. Uninoculated controls (n = 100) were negative for mcr-1 following enrichment.
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| 2. |
- Dierikx, Cindy, et al.
(författare)
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A European multicenter evaluation study to investigate the performance on commercially available selective agar plates for the detection of carbapenemase producing Enterobacteriaceae
- 2022
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Ingår i: Journal of Microbiological Methods. - : Elsevier. - 0167-7012 .- 1872-8359. ; 193
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Tidskriftsartikel (refereegranskat)abstract
- The European Food Safety Authority (EFSA) advised to prioritize monitoring carbapenemase producing Enterobacteriaceae (CPE) in food producing animals. Therefore, this study evaluated the performance of different commercially available selective agars for the detection of CPE using spiked pig caecal and turkey meat samples and the proposed EFSA cultivation protocol. Eleven laboratories from nine countries received eight samples (four caecal and four meat samples). For each matrix, three samples contained approximately 100 CFU/g CPE, and one sample lacked CPE. After overnight enrichment in buffered peptone water, broths were spread upon Brilliance™ CRE Agar (1), CHROMID® CARBA (2), CHROMagar™ mSuperCARBA™ (3), Chromatic™ CRE (4), CHROMID® OXA-48 (5) and Chromatic™ OXA-48 (6). From plates with suspected growth, one to three colonies were selected for species identification, confirmation of carbapenem resistance and detection of carbapenemase encoding genes, by methods available at participating laboratories. Of the eleven participating laboratories, seven reported species identification, susceptibility tests and genotyping on isolates from all selective agar plates. Agars 2, 4 and 5 performed best, with 100% sensitivity. For agar 3, a sensitivity of 96% was recorded, while agar 1 and 6 performed with 75% and 43% sensitivity, respectively. More background flora was noticed for turkey meat samples than pig caecal samples. Based on this limited set of samples, most commercially available agars performed adequately. The results indicate, however, that OXA-48-like and non-OXA-48-like producers perform very differently, and one should consider which CPE strains are of interest to culture when choosing agar type.
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| 3. |
- Fatima, Masoom, et al.
(författare)
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Application of novel bacterial consortium for biodegradation of aromatic amine 2-ABS using response surface methodology
- 2020
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 174
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Tidskriftsartikel (refereegranskat)abstract
- There is a strong need to develop purification methods for textile industrial wastewater containing toxic azo dyes. The reductive cleavage of azo dyes can be made by anaerobic bacteria, but the products of aromatic amines require an aerobic process. In this study a novel bacterial dye degrading consortium (DDC) of five isolated strains identified with 16S rRNA sequence: Proteus mirabilis (KR732288), Bacillus anthracis (KR732289), Enterobacter hormaechei (KR732290), Pseudomonas aeruginosa (KR732293) and Serratia rubidaea (KR732296) were used to aerobically decompose metabolite 2-aminobenxenesulfonic acid (2-ABS), as a model compound. The effect of three variables: temperature (28-42 degrees C), pH (5.0-8.0) and initial concentration of 2-ABS (5-40 ppm) was investigated in terms of degradation and chemical oxygen demand (COD) removal. Central composite design matrixand response surface methodology (RSM) were used for experimental design to evaluate theinteraction of the three process variables. The results show that up to 95% degradation and COD 90% removal are possible at optimal values of 32.4 ppm 2-ABS, pH 6.6 and a temperature of 35.7 degrees C. The theoretical response variables predicted by the developed RSM model was supported the experimental results. The optimized degradation of 2-ABS and COD removal were further confirmed by UV-HPLC analysis.
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| 4. |
- Forsgren, Eva
(författare)
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An international inter-laboratory study on Nosema spp. spore detection and quantification through microscopic examination of crushed honey bee abdomens
- 2021
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 184
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Tidskriftsartikel (refereegranskat)abstract
- Nosemosis is a microsporidian disease causing mortality and weakening of honey bee colonies, especially in the event of co-exposure to other sources of stress. As a result, the disease is regulated in some countries. Reliable and harmonised diagnosis is crucial to ensure the quality of surveillance and research results. For this reason, the first European Interlaboratory Comparison (ILC) was organised in 2017 in order to assess both the methods and the results obtained by National Reference Laboratories (NRLs) in counting Nosema spp. spores by microscopy. Implementing their own routine conditions of analysis, the 23 participants were asked to perform an assay on & nbsp;a & nbsp;panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study concluded that using microscopy to detect and quantify spores of Nosema spp. was reliable and valid.panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study concluded
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| 5. |
- Jagadish, Anupama, et al.
(författare)
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Development and optimization of a TaqMan assay for Nosema bombycis, causative agent of pebrine disease in Bombyx mori silkworm, based on the β-tubulin gene
- 2021
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 186
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Tidskriftsartikel (refereegranskat)abstract
- “Pébrine” is a devastating disease of Bombyx mori silkworms that is highly contagious and can completely destroy an entire crop of silkworms and is thus a serious threat for the viability and profitability of sericulture. The disease is most commonly attributed to microsporidians of the genus Nosema, which are obligate intracellular parasites that are transmitted through spores. Nosema infections in silkworms are diagnosed primarily through light microscopy, which is labour intensive and less reliable, sensitive, and specific than PCR-based techniques. Here, we present the development and optimization of a new TaqMan based assay targeting the β-tubulin gene in the pébrine disease causing agent Nosema bombycis in silkworms. The assay displayed excellent quantification linearity over multiple orders of magnitude of target amounts and a limit of detection (LOD) of 6.9 × 102 copies of target per reaction. The method is highly specific to N. bombycis with no cross-reactivity to other Nosema species commonly infecting wild silkworms. This specificity was due to three nucleotides in the probe-binding region unique to N. bombycis. The assay demonstrated a high reliability with a Coefficient of variation (CV) <5% for both intra-assay and inter-assay variability. The assay was used to trace experimental N. bombycis infection of silkworm larvae, in the fat body, midgut and ovary tissues, through pupation and metamorphosis to the emerging female moth, and her larval off-spring, confirming the vertical transmission of N. bombycis in silkworms. The TaqMan assay revealed a gradual increase in infection levels in the post-infection samples. The assay is reliable and simple to implement and can be a suitable complement to microscopy for routine diagnostics and surveillance in silkworm egg production centres with appropriate infrastructure.
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| 6. |
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| 7. |
- Sjöberg, Fei, et al.
(författare)
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Are all faecal bacteria detected with equal efficiency? A study using next-generation sequencing and quantitative culture of infants' faecal samples
- 2020
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 177
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Tidskriftsartikel (refereegranskat)abstract
- Background: Many species of intestinal bacteria are present in moderate numbers in the faecal microbiota, which is dominated by obligate anaerobes. Little is known regarding the detection sensitivity of next-generation sequencing for these microbes in samples of complex microbiota. Methods: Twenty stool samples from six healthy infants, who were followed from 1 week to 1 year of age, were previously cultured quantitatively for total population counts, as well as for counts of relevant facultative bacteria and a limited selection of obligate anaerobes that are prevalent in the neonatal microbiota. The same samples were analysed by Next-generation sequencing (NGS, pyrosequencing) of the 16S rRNA gene (V1–V3 regions; average read length, 500 nucleotides; average number of reads per sample, 30,000). We used the bacterial culture data to determine the lowest bacterial populations that could be detected by NGS. Different DNA extraction kits (QIAamp DNA Stool Mini, ZR Faecal DNA MiniPrep, and PowerSoil DNA Isolation) were compared for efficacy in extracting DNA from Gram-negative and Gram-positive Type strains. Results: NGS yielded one read per 106 CFU/g faeces of the Gram-negative commensal gut bacteria Bacteroides and Enterobacteriaceae, but only one read per 108 CFU/g faeces of Gram-positive bifidobacteria. The Gram-positive facultative bacteria Enterococcus was often undetectable by DNA-based methods despite being present at >106 CFU/g faeces. The DNA extraction kits tested varied considerably in their ability to extract DNA from bacterial samples, and showed considerably lower efficacies in extracting DNA from Gram-positive than from Gram-negative bacteria. Conclusions: NGS has lower sensitivity for detecting Gram-positive bacteria than Gram-negative bacteria, due at least in part to inefficient extraction of DNA from Gram-positive bacteria. Therefore, enzymatic lysis may enhance the yield of DNA and increase the sensitivity of NGS methods for Gram-positive bacteria, and the inclusion of positive and negative controls during DNA extraction is indicated for validation purposes. The differential extraction of DNA from bacterial samples by different DNA extraction kits may limit comparability between studies on the gut microbiota. Finally, quantitative culture methods detect certain bacteria with greater sensitivity than NGS techniques, and thus culture- and DNA-based methods can be used in tandem to define the complex composition of the gut microbiota with greater accuracy.
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| 8. |
- Sune, Dan, et al.
(författare)
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Optimization of 16S rRNA gene analysis for use in the diagnostic clinical microbiology service
- 2020
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Ingår i: Journal of Microbiological Methods. - : ELSEVIER. - 0167-7012 .- 1872-8359. ; 170
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Tidskriftsartikel (refereegranskat)abstract
- Broad-range amplification and sequencing of the 16S rRNA gene, directly from clinical samples, is a method that potentially allows detection of any cultivable or non-cultivable bacteria. However, the method is prone to false positive results due to PCR contamination. Another concern is the human DNA abundance compared to bacterial DNA in samples from sterile sites. Those factors may decrease the sensitivity and specificity of the assay and can complicate the analysis and interpretation of the results. The objective of this prospective study was to try to avoid the most common pitfalls, mentioned above, and develop a molecular 16S assay with a high clinical sensitivity and specificity. Fifty-six consecutive tissue samples from patients with suspected deep infections were extracted by 3 different DNA-extraction methods; two based on a principle of bacterial DNA enrichment, and one conventional DNA extraction method. We compared three primer pairs, including both conventional and DPO principle, targeting different variable regions of the 16S rRNA gene. Results from routine tissue culture were used as reference. Clinical data was recorded from patient charts and analyzed in parallel. Of a total of 56 samples, collected from 39 patients, 70% (39 samples) were assessed as true infections by analysis of clinical data. Bacterial enrichment extraction increased sensitivity from 54% to 72%. The 2 sets of primer pairs defining region V1-V3 and V3-V4, showed similar sensitivity, but DPO-primers resulted in better specificity, i.e. less contaminations. The primer pairs covering V1-V8 show significantly lower sensitivity (p amp;lt; .001) than V1-V3 and V3-V4. Optimizing extraction protocols and choice of primers can increase the sensitivity and specificity of a molecular 16S-analysis, rendering a valuable complement to tissue culture.
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| 9. |
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| 10. |
- Jansson, Linda, et al.
(författare)
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Impact of swab material on microbial surface sampling
- 2020
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012. ; 176
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Tidskriftsartikel (refereegranskat)abstract
- Efficient microbial sampling from surfaces for subsequent detection and quantification is crucial in fields such as food safety and hygiene monitoring. Cotton swabs are traditionally used for sample collection, but today there are numerous swabs of alternative material and different sizes available. Recovery efficiencies of swabs for different applications have been compared in several studies. However, the results are often contradictory. We have compared 15 different swabs made of cotton (n = 5), flocked nylon (n = 3) and foam (n = 7), for sampling of Listeria monocytogenes and mengovirus on small (4 cm2) and large (100 cm2) areas of window glass, ridged plastic and absorbing wood. Molecular quantification methods (qPCR and RT-qPCR) were applied, and all sampling and sample processing were standardized. Specific swabs gave higher DNA/RNA yields than others, depending on both the surface characteristics and the collected target. The highest DNA yields were achieved by applying Selefa or Puritan cotton swabs for Listeria sampling on 4 cm2 areas of window glass and ridged plastic. Certain foam swabs (Critical swab with medium head and Macrofoam) gave the highest yields when sampling Listeria on 4 cm2 areas of wood and on 100 cm2 areas of ridged plastic and wood. Most foam swabs, and especially Sigma Virocult, were advantageous for virus sampling, regardless of surface. Nylon-flocked swabs showed poor recovery regardless of surface characteristics. The recovery varied substantially between swabs made of the same material, suggesting that a single swab may not be representative for a certain swab material.
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