Janet Martha Blatny

Characterization of airborne bacteria at an underground subway station

ABSTRACT The reliable detection of airborne biological threat agents depends on several factors, including the performance criteria of the detector and its operational environment. One step in improving the detectors performance is to increase our knowledge of the biological aerosol background in potential operational environments. Subway stations are enclosed public environments, which may be regarded as potential targets for incidents involving biological threat agents. In this study, the airborne bacterial community at a subway station in Norway was characterized (concentration level, diversity, and virulence- and survival-associated properties). In addition, a SASS 3100 high-volume air sampler and a matrix-assisted laser desorption ionization–time of flight mass spectrometry-based isolate screening procedure was used for these studies. The daytime level of airborne bacteria at the station was higher than the nighttime and outdoor levels, and the relative bacterial spore number was higher in outdoor air than at the station. The bacterial content, particle concentration, and size distribution were stable within each environment throughout the study (May to September 2010). The majority of the airborne bacteria belonged to the genera Bacillus, Micrococcus, and Staphylococcus, but a total of 37 different genera were identified in the air. These results suggest that anthropogenic sources are major contributors to airborne bacteria at subway stations and that such airborne communities could harbor virulence- and survival-associated properties of potential relevance for biological detection and surveillance, as well as for public health. Our findings also contribute to the development of realistic testing and evaluation schemes for biological detection/surveillance systems by providing information that can be used to mimic real-life operational airborne environments in controlled aerosol test chambers.

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.

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.

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.

L-alanine-induced germination in Bacillus licheniformis -the impact of native gerA sequences

BackgroundL-alanine, acting through the GerA receptor, was recently found to be an efficient germinant in Bacillus licheniformis ATCC14580/DSM13.ResultsIn this study, we show that several of 46 examined B. licheniformis strains germinate remarkably slower than the type strain when exposed to L-alanine. These strains are not necessarily closely related, as determined by MLST (multi-locus sequence typing). Three of the slow-germinating strains were further examined in order to see whether nucleotide substitutions in the gerA sequences were responsible for the slow L-alanine germination. This was performed by complementing the transformable type strain derivate MW3ΔgerAA with gerA variants from the three slow-germinating strains; NVH1032, NVH1112 and NVH800.ConclusionsA wide selection of B. licheniformis strains was evaluated for L-alanine-induced germination efficiency. Our results show that gerA substitutions could only partially explain why spores of some B. licheniformis strains responded slower than others in the presence of L-alanine.

Detecting and Responding to Bioterrorism

rickettsiae, fungi, viruses, or toxins with the intent to incapacitate or kill man, or to destroy livestock, crops, or food. The use of biological threat agents in biological warfare has a long history, but the recent use of anthrax spores in bioterrorism has urged the need for responding to such threats. The design of efficient and reliable detection and identification systems is a part of bioterrorism preparedness and response. Classical microbiology, immunoassays, and nucleic acid-based methods including molecular forensics are laboratory approaches for detecting and identifying various biological threat agents. These are supplementary methods needed for verification. However, the analysis results depend on the sample collection and handling. This chapter briefly summarizes some biological warfare and bioterrorism events, the threat posed by bioterrorism. Also, various methods and systems suitable for detection and identification of biological

Tracking Legionella in air generated from a biological treatment plant - A case study of the outbreak of legionellosis in Norway-

Two outbreaks of legionellosis occurred in the Sarpsborg/Fredrikstad region southeast of Norway in 2005 and 2008 where more than 60 exposed individuals were infected and 10 case patients died. The air scrubber at Borregaard, a wood-based chemical factory, was identified as the outbreak source. High concentration levels of Legionella species, including the etiological agent L. pneumophila SG1 was found in the aeration ponds, which belongs to Borregaards biological treatment plant. Results showed that these ponds were able to generate Legionella-containing aerosols that were transported by the wind as such aerosols were measured up to 200 meters downwind of the pond. Our studies did not detect L. pneumophila SG1 isolates, only L. pneumophila SG4 during the air sampling measurement campaign. Furthermore, the operational conditions of the air scrubber proved to be harsh for Legionella growth as the outbreak L. pneumophila strains were not able to grow at 45ºC and pH8 (conditions during the outbreaks). These results, together, lead us to suggest that the aeration pond should be regarded as the primary amplifier and disseminator of Legionella and L. pneumophila and thereby most likely being the outbreak source.