Jaran Strand Olsen

Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR

A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence. The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation. The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria. It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication. The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth. The 4-h-old culture was also more sensitive for lysis caused by heating. The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication. However, the yield of target DNA from E. coli rapidly decreased after 50 s of sonication due to degradation of DNA. Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B. cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR. The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.

Evaluation of a highly discriminating multiplex multi-locus variable-number of tandem-repeats (MLVA) analysis for Vibrio cholerae

Vibrio cholerae is the etiological agent of cholera and may be used in bioterror actions due to the easiness of its dissemination, and the public fear for acquiring the cholera disease. A simple and highly discriminating method for connecting clinical and environmental isolates of V. cholerae is needed in microbial forensics. Twelve different loci containing variable numbers of tandem-repeats (VNTRs) were evaluated in which six loci were polymorphic. Two multiplex reactions containing PCR primers targeting these six VNTRs resulted in successful DNA amplification of 142 various environmental and clinical V. cholerae isolates. The genetic distribution inside the V. cholerae strain collection was used to evaluate the discriminating power (Simpsons Diversity Index=0.99) of this new MLVA analysis, showing that the assay have a potential to differentiate between various strains, but also to identify those isolates which are collected from a common V. cholerae outbreak. This work has established a rapid and highly discriminating MLVA assay useful for track back analyses and/or forensic studies of V. cholerae infections.

A multiplatform real-time polymerase chain reaction detection assay for Vibrio cholerae

We report a multiplatform real-time polymerase chain reaction methodology based on genes encoding for the regulatory toxR activator and enterotoxin A protein to determine enterotoxigenic Vibrio cholerae types from other vibrios. This assay, which was successfully validated on a collection of 87 bacterial strains, including 63 representatives of V. cholerae and 8 noncholera vibrios provides a rapid tool for detection and identification of cholera.

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Detection of bioterror agents in air samples using real‐time PCR

Aims:  To use real‐time PCR for the detection of bacterial bioterror agents in a liquid air sample containing potential airborne interferences, including bacteria, without the need for DNA extraction.

Analysis of the genetic distribution among members of Clostridium botulinum group I using a novel multilocus sequence typing (MLST) assay.

Clostridium botulinum is the etiological agent of botulism. Due to food-borne poisoning and the potential use of the extremely toxic botulinum neurotoxin (BoNT) from C. botulinum in bioterror or biocrime related actions, reliable high resolution typing methods for discriminating C. botulinum strains are needed. Partial sequencing of the adk, atpH, gyrB, proC, rpoD and spo0A genes from 51 various C. botulinum/sporogenes isolates was performed, resulting in 37 different sequence types (STs). Analysis of the sequence data revealed a genetic distribution in five larger clusters with a loose correlation to the BoNT serotypes. The developed MLST assay had a slightly lower resolution ability when compared to the MLVA (multilocus variable number of tandem repeat analysis), but the two methods resulted in similar subclusters of the strains possessing the BoNT serotypes A, B and F. The current work presents the development of a novel MLST assay useful for genotyping C. botulinum related to basic phylogenetic research and trace-back analysis in microbial forensic studies.

Genotyping of B. licheniformis based on a novel multi-locus sequence typing (MLST) scheme

BackgroundBacillus licheniformis has for many years been used in the industrial production of enzymes, antibiotics and detergents. However, as a producer of dormant heat-resistant endospores B. licheniformis might contaminate semi-preserved foods. The aim of this study was to establish a robust and novel genotyping scheme for B. licheniformis in order to reveal the evolutionary history of 53 strains of this species. Furthermore, the genotyping scheme was also investigated for its use to detect food-contaminating strains.ResultsA multi-locus sequence typing (MLST) scheme, based on the sequence of six house-keeping genes (adk, ccpA, recF, rpoB, spo0A and sucC) of 53 B. licheniformis strains from different sources was established. The result of the MLST analysis supported previous findings of two different subgroups (lineages) within this species, named “A” and “B” Statistical analysis of the MLST data indicated a higher rate of recombination within group “A”. Food isolates were widely dispersed in the MLST tree and could not be distinguished from the other strains. However, the food contaminating strain B. licheniformis NVH1032, represented by a unique sequence type (ST8), was distantly related to all other strains.ConclusionsIn this study, a novel and robust genotyping scheme for B. licheniformis was established, separating the species into two subgroups. This scheme could be used for further studies of evolution and population genetics in B. licheniformis.

Genetic characterization of trh positive Vibrio spp. isolated from Norway

The thermostable direct hemolysin (TDH) and/or TDH-related hemolysin (TRH) genes are carried by most virulent Vibrio parahaemolyticus serovars. In Norway, trh+ V. parahaemolyticus constitute 4.4 and 4.5% of the total number of V. parahaemolyticus isolated from blue mussel (Mytilus edulis) and water, respectively. The trh gene is located in a region close to the gene cluster for urease production (ure). This region was characterized in V. parahaemolyticus strain TH3996 and it was found that a nickel transport operon (nik) was located between the first gene (ureR) and the rest of the ure cluster genes. The organization of the trh-ureR-nik-ure gene cluster in the Norwegian trh+ isolates was unknown. In this study, we explore the gene organization within the trh-ureR-nik-ure cluster for these isolates. PCR analyses revealed that the genes within the trh-ureR-nik-ure gene cluster of Norwegian trh+ isolates were organized in a similar fashion as reported previously for TH33996. Additionally, the phylogenetic relationship among these trh+ isolates was investigated using Multilocus Sequence Typing (MLST). Analysis by MLST or ureR-trh sequences generated two different phylogenetic trees for the same strains analyzed, suggesting that ureR-trh genes have been acquired at different times in Norwegian V. parahaemolyticus isolates. MLST results revealed that some pathogenic and non-pathogenic V. parahaemolyticus isolates in Norway appear to be highly genetically related.

Preliminary validation of real-time PCR assays for the identification of Yersinia pestis

Abstract Background: Yersinia pestis (Y. pestis) is a zoonotic bacterium mainly circulating among rodents and their fleas. Transmission to humans can cause bubonic, pneumonic or septicemic plague with a high case-fatality rate. Therefore, rapid and reliable diagnostic tools are crucial. The objective of this study was to assess the inter-laboratory reproducibility of in-house developed real-time PCR assays for the identification of Y. pestis. Methods: A total of four samples of quantified Y. pestis DNA and two blank samples were sent blinded to 14 laboratories. To standardize the procedures, oligonucleotides were provided and the same instrument platform and a commercial mastermix were used. The participants were requested to report their results including cycle threshold and melting temperature values. Results: All participating laboratories were able to perform the real-time PCR assays according to the protocols provided and identified the samples containing Y. pestis DNA correctly. Significant differences between the reference laboratory and participating laboratories were observed in cycle threshold values and melting temperatures. This, however, did not adversely affect the interpretation of results. Conclusions: Our real-time PCR system proved to be highly reproducible and has the potential of complementing the diagnostic tools for rapid identification of Y. pestis isolates. Further steps of validation are needed to determine diagnostic accuracy and predictive values with clinical samples. Clin Chem Lab Med 2008;46:1239–44.

OmpU as a biomarker for rapid discrimination between toxigenic and epidemic Vibrio cholerae O1/O139 and non-epidemic Vibrio cholerae in a modified MALDI-TOF MS assay

BackgroundCholera is an acute diarrheal disease caused by Vibrio cholerae. Outbreaks are caused by a genetically homogenous group of strains from serogroup O1 or O139 that are able to produce the cholera toxin. Rapid detection and identification of these epidemic strains is essential for an effective response to cholera outbreaks.ResultsThe use of ferulic acid as a matrix in a new MALDI-TOF MS assay increased the measurable mass range of existing MALDI-TOF MS protocols for bacterial identification. The assay enabled rapid discrimination between epidemic V. cholerae O1/O139 strains and other less pathogenic V. cholerae strains. OmpU, an outer membrane protein whose amino acid sequence is highly conserved among epidemic strains of V. cholerae, appeared as a discriminatory marker in the novel MALDI-TOF MS assay.ConclusionsThe extended mass range of MALDI-TOF MS measurements obtained by using ferulic acid improved the screening for biomarkers in complex protein mixtures. Differences in the mass of abundant homologous proteins due to variation in amino acid sequences can rapidly be examined in multiple samples. Here, a rapid MALDI-TOF MS assay was developed that could discriminate between epidemic O1/O139 strains and other less pathogenic V. cholerae strains based on differences in mass of the OmpU protein. It appeared that the amino acid sequence of OmpU from epidemic V. cholerae O1/O139 strains is unique and highly conserved.