Gunnar Skogan

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.

Temporal Variability of the Bioaerosol Background at a Subway Station: Concentration Level, Size Distribution and Diversity of Airborne Bacteria

ABSTRACT Naturally occurring bioaerosol environments may present a challenge to biological detection-identification-monitoring (BIODIM) systems aiming at rapid and reliable warning of bioterrorism incidents. One way to improve the operational performance of BIODIM systems is to increase our understanding of relevant bioaerosol backgrounds. Subway stations are enclosed public environments which may be regarded as potential bioterrorism targets. This study provides novel information concerning the temporal variability of the concentration level, size distribution, and diversity of airborne bacteria in a Norwegian subway station. Three different air samplers were used during a 72-h sampling campaign in February 2011. The results suggested that the airborne bacterial environment was stable between days and seasons, while the intraday variability was found to be substantial, although often following a consistent diurnal pattern. The bacterial levels ranged from not detected to 103 CFU m−3 and generally showed increased levels during the daytime compared to the nighttime levels, as well as during rush hours compared to non-rush hours. The airborne bacterial levels showed rapid temporal variation (up to 270-fold) on some occasions, both consistent and inconsistent with the diurnal profile. Airborne bacterium-containing particles were distributed between different sizes for particles of >1.1 μm, although ∼50% were between 1.1 and 3.3 μm. Anthropogenic activities (mainly passengers) were demonstrated as major sources of airborne bacteria and predominantly contributed 1.1- to 3.3-μm bacterium-containing particles. Our findings contribute to the development of realistic testing and evaluation schemes for BIODIM equipment by providing information that may be used to simulate operational bioaerosol backgrounds during controlled aerosol chamber-based challenge tests with biological threat agents.

Comparative Testing and Evaluation of Nine Different Air Samplers: End-to-End Sampling Efficiencies as Specific Performance Measurements for Bioaerosol Applications

Accurate exposure assessments are needed to evaluate health hazards caused by airborne microorganisms and require air samplers that efficiently capture representative samples. This highlights the need for samplers with well-defined performance characteristics. While generic aerosol performance measurements are fundamental to evaluate/compare samplers, the added complexity caused by the diversity of microorganisms, especially in combination with cultivation-based analysis methods, may render such measurements inadequate to assess suitability for bioaerosols. Specific performance measurements that take into account the end-to-end sampling process, targeted bioaerosol and analysis method could help guide selection of air samplers. Nine different samplers (impactors/impingers/cyclones/ electrostatic precipitators/filtration samplers) were subjected to comparative performance testing in this work. Their end-to-end cultivation-based biological sampling efficiencies (BSEs) and PCR-/microscopy-based physical sampling efficiencies (PSEs) relative to a reference sampler (BioSampler) were determined for gram-negative and gram-positive vegetative bacteria, bacterial spores, and viruses. Significant differences were revealed among the samplers and shown to depend on the bioaerosols stress–sensitivity and particle size. Samplers employing dry collection had lower BSEs for stress-sensitive bioaerosols than wet collection methods, while nonfilter-based samplers showed reduced PSEs for 1 μm compared to 4 μm bioaerosols. Several samplers were shown to underestimate bioaerosol concentration levels relative to the BioSampler due to having lower sampling efficiencies, although they generally obtained samples that were more concentrated due to having higher concentration factors. Our work may help increase user awareness about important performance criteria for bioaerosol sampling, which could contribute to methodological harmonization/standardization and result in more reliable exposure assessments for airborne pathogens and other bioaerosols of interest. Copyright 2014 American Association for Aerosol Research

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.

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.

Standoff detection of biological agents using laser induced fluorescence—a comparison of 294 nm and 355 nm excitation wavelengths

Standoff detection measuring the fluorescence spectra of seven different biological agents excited by 294 nm as well as 355 nm wavelength laser pulses has been undertaken. The biological warfare agent simulants were released in a semi-closed aerosol chamber at 210 m standoff distance and excited by light at either of the two wavelengths using the same instrument. Significant differences in several of the agents’ fluorescence response were seen at the two wavelengths. The anthrax simulants’ fluorescence responses were almost an order of magnitude stronger at the shorter wavelength excitation. However, most importantly, the fluorescence spectra were significantly more dissimilar at 294 nm than at 355 nm excitation with ~7 nm spectral resolution. This indicates that classification of the substances should be possible with a lower error rate for standoff detection using 294 nm rather than 355 nm excitation wavelength, or even better, utilizing both.

Aerobiological stability of different species of Gram-negative bacteria, including well-known biothreat simulants, in single-cell particles and cell clusters of different composition

ABSTRACT The ability to perform controlled experiments with bioaerosols is a fundamental enabler of many bioaerosol research disciplines. A practical alternative to using hazardous biothreat agents, e.g., for detection equipment development and testing, involves using appropriate model organisms (simulants). Several species of Gram-negative bacteria have been used or proposed as biothreat simulants. However, the appropriateness of different bacterial genera, species, and strains as simulants is still debated. Here, we report aerobiological stability characteristics of four species of Gram-negative bacteria (Pantoea agglomerans, Serratia marcescens, Escherichia coli, and Xanthomonas arboricola) in single-cell particles and cell clusters produced using four spray liquids (H2O, phosphate-buffered saline[PBS], spent culture medium[SCM], and a SCM-PBS mixture). E. coli showed higher stability in cell clusters from all spray liquids than the other species, but it showed similar or lower stability in single-cell particles. The overall stability was higher in cell clusters than in single-cell particles. The highest overall stability was observed for bioaerosols produced using SCM-containing spray liquids. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. Taken together, the results highlight the importance of careful and informed selection of Gram-negative bacterial biothreat simulants and also the accompanying particle size and composition. The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research. IMPORTANCE The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research. Taken together, the results highlight the importance of careful and informed selection of Gram-negative bacterial biothreat simulants and also the accompanying particle size and composition. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns.