Gamma Irradiation Can Be Used To Inactivate
            <i>Bacillus anthracis</i>
            Spores without Compromising the Sensitivity of Diagnostic Assays

Dauphin, Leslie A.; Newton, Bruce R.; Rasmussen, Max V.; Meyer, Richard F.; Bowen, Michael D.

doi:10.1128/aem.00557-08

Cited by 36 publications

(30 citation statements)

References 43 publications

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“…High-dose gamma irradiation is known in the literature as a method suitable for reliably inactivating bacterial pathogens (42,43) while leaving the primary protein structures basically intact. Our comparative measurements of pathogenic and nonpathogenic microbial strains essentially confirmed the data in the literature: identification was successful after high-dose gamma irradiation, but irradiation resulted in slightly lower BioTyper log score values (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Identification of Highly Pathogenic Microorganisms by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Results of an Interlaboratory Ring Trial

et al. 2015

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mIn the case of a release of highly pathogenic bacteria (HPB), there is an urgent need for rapid, accurate, and reliable diagnostics. MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive technique that is becoming increasingly important in microbiological diagnostics to complement classical microbiology, PCR, and genotyping of HPB. In the present study, the results of a joint exercise with 11 partner institutions from nine European countries are presented. In this exercise, 10 distinct microbial samples, among them five HPB, Bacillus anthracis, Brucella canis, Burkholderia mallei, Burkholderia pseudomallei, and Yersinia pestis, were characterized under blinded conditions. Microbial strains were inactivated by high-dose gamma irradiation before shipment. Preparatory investigations ensured that this type of inactivation induced only subtle spectral changes with negligible influence on the quality of the diagnosis. Furthermore, pilot tests on nonpathogenic strains were systematically conducted to ensure the suitability of sample preparation and to optimize and standardize the workflow for microbial identification. The analysis of the microbial mass spectra was carried out by the individual laboratories on the basis of spectral libraries available on site. All mass spectra were also tested against an in-house HPB library at the Robert Koch Institute (RKI). The averaged identification accuracy was 77% in the first case and improved to >93% when the spectral diagnoses were obtained on the basis of the RKI library. The compilation of complete and comprehensive databases with spectra from a broad strain collection is therefore considered of paramount importance for accurate microbial identification. Highly pathogenic bacteria (HPB) are risk group 3 bacteria, which are defined as biological agents that can cause severe human disease and present a serious hazard to health care workers. This may present a risk of spreading to the community, but there is usually effective prophylaxis or treatment available (1). To this group belong bacteria such as Bacillus anthracis, Francisella tularensis subsp. tularensis (type A), Yersinia pestis, species of the Brucella melitensis group, Burkholderia mallei, and Burkholderia pseudomallei. HPB have the potential to be used in bioterrorist attacks (2, 3). The Centers for Disease Control and Prevention (CDC, Atlanta, GA) have classified B. anthracis, F. tularensis, and Y. pestis as category A and Brucella species, B. mallei, B. pseudomallei, and Coxiella burnetii as category B, comprising the main pathogens of concern for use in bioterrorist attacks (4). These pathogens may cause anthrax, tularemia, plague, brucellosis, glanders, melioidosis, and Q fever, respectively. In most parts of the world, the natural prevalence of these agents is low, even though some of these agents cause outbreaks in human and animal populations from time to time (5-8). The intentional release of these agents, however, can result in severe public health consequences, as was shown in the U...

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Section: Resultsmentioning

confidence: 99%

Identification of Highly Pathogenic Microorganisms by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Results of an Interlaboratory Ring Trial

et al. 2015

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“…In spite of the known effects of high-dose irradiation on spore viability (16)(17)(18)(19)(20), information about its impact on WGS methods or data quality is limited. Several works have demonstrated the utility of irradiated material for use in immunoassay and PCRbased detection (15)(16)(17), and other investigators have reported the phylogenetic classification of Yersinia pestis strains from highly degraded samples originating from the period of the Black Death (21), which suffer DNA lesions and fragmentation similar to those suffered by irradiated samples.…”

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confidence: 99%

“…Several works have demonstrated the utility of irradiated material for use in immunoassay and PCRbased detection (15)(16)(17), and other investigators have reported the phylogenetic classification of Yersinia pestis strains from highly degraded samples originating from the period of the Black Death (21), which suffer DNA lesions and fragmentation similar to those suffered by irradiated samples. However, little literature describing the effect of irradiation on DNA within vegetative cells, spores, or naked DNA was available prior to the commencement of this study.…”

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confidence: 99%

Whole-Genome Sequencing in Microbial Forensic Analysis of Gamma-Irradiated Microbial Materials

Broomall¹,

Ichou

Krepps

et al. 2016

Appl Environ Microbiol

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Effective microbial forensic analysis of materials used in a potential biological attack requires robust methods of morphological and genetic characterization of the attack materials in order to enable the attribution of the materials to potential sources and to exclude other potential sources. The genetic homogeneity and potential intersample variability of many of the category A to C bioterrorism agents offer a particular challenge to the generation of attributive signatures, potentially requiring whole-genome or proteomic approaches to be utilized. Currently, irradiation of mail is standard practice at several government facilities judged to be at particularly high risk. Thus, initial forensic signatures would need to be recovered from inactivated (nonviable) material. In the study described in this report, we determined the effects of high-dose gamma irradiation on forensic markers of bacterial biothreat agent surrogate organisms with a particular emphasis on the suitability of genomic DNA (gDNA) recovered from such sources as a template for whole-genome analysis. While irradiation of spores and vegetative cells affected the retention of Gram and spore stains and sheared gDNA into small fragments, we found that irradiated material could be utilized to generate accurate whole-genome sequence data on the Illumina and Roche 454 sequencing platforms.

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“…The cell pellets were resuspended in 5 ml of sterile phosphate-buffered saline (PBS; 0.01 M, pH 7.4), and the total culture volume was divided into two equal aliquots designated harvest A and harvest B. Harvest A was immediately used as an inoculum to spike the swabs, numbers of CFU/ml were quantified on TSAB plates using standard procedures (9), and the cells were examined by staining and light microscopy (see below). Harvest B underwent a heat-shock treatment (30 min at 65°C) to verify that the inoculum was spore free.…”

Section: Scientific Ethicsmentioning

confidence: 99%

“…This is a highly sensitive and specific assay that was validated for detection of B. anthracis and was successfully used to test hundreds of samples during the 2001 bioterrorism-associated anthrax outbreak. The assay includes three primer and probe sets which target the B. anthracis chromosome and the pXO1 and pXO2 plasmids and has been described in previous reports (9,16). PCR was carried out in triplicate 25-l volumes.…”

Section: Scientific Ethicsmentioning

confidence: 99%

Swab Protocol for Rapid Laboratory Diagnosis of Cutaneous Anthrax

et al. 2012

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The clinical laboratory diagnosis of cutaneous anthrax is generally established by conventional microbiological methods, such as culture and directly straining smears of clinical specimens. However, these methods rely on recovery of viable Bacillus anthracis cells from swabs of cutaneous lesions and often yield negative results. This study developed a rapid protocol for detection of B. anthracis on clinical swabs. Three types of swabs, flocked-nylon, rayon, and polyester, were evaluated by 3 extraction methods, the swab extraction tube system (SETS), sonication, and vortex. Swabs were spiked with virulent B. anthracis cells, and the methods were compared for their efficiency over time by culture and real-time PCR. Viability testing indicated that the SETS yielded greater recovery of B. anthracis from 1-day-old swabs; however, reduced viability was consistent for the 3 extraction methods after 7 days and nonviability was consistent by 28 days. Real-time PCR analysis showed that the PCR amplification was not impacted by time for any swab extraction method and that the SETS method provided the lowest limit of detection. When evaluated using lesion swabs from cutaneous anthrax outbreaks, the SETS yielded culture-negative, PCR-positive results. This study demonstrated that swab extraction methods differ in their efficiency of recovery of viable B. anthracis cells. Furthermore, the results indicated that culture is not reliable for isolation of B. anthracis from swabs at >7 days. Thus, we recommend the use of the SETS method with subsequent testing by culture and real-time PCR for diagnosis of cutaneous anthrax from clinical swabs of cutaneous lesions.

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Gamma Irradiation Can Be Used To Inactivate Bacillus anthracis Spores without Compromising the Sensitivity of Diagnostic Assays

Cited by 36 publications

References 43 publications

Identification of Highly Pathogenic Microorganisms by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Results of an Interlaboratory Ring Trial

Identification of Highly Pathogenic Microorganisms by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Results of an Interlaboratory Ring Trial

Whole-Genome Sequencing in Microbial Forensic Analysis of Gamma-Irradiated Microbial Materials

Swab Protocol for Rapid Laboratory Diagnosis of Cutaneous Anthrax

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