Lab researchers examine impact of sample-collection methods on pathogen sample recovery

Microscopic analysis of B. anthracis spores

Mar. 18, 2010—Elizabeth Hong-Geller, Yulin Shou, and Babs Marrone of Biosecurity and Public Health; Yolanda Valdez and John Dunbar of Bioenergy and Environmental Science; and Thomas Yoshida of Chemical Diagnostics and Engineering have evaluated sample recovery efficiencies for Bacillus anthracis and Yersinia pesti, the causative agents of anthrax and plague. Increasing the number of bacteria removed from a surface, the "efficiency of recovery" enhances the likelihood that there will be enough sample for analysis. The research team is trying to determine the impact of different sample collection methods on the recovery of pathogen signatures from nonporous surfaces.

Bioforensic methods for rapid and reliable detection of biothreat agents are key capabilities for evidence collection and assessment of surface contamination after bioterrorism attacks. Following the dissemination of B. anthracis spores through the U.S. Postal Service in 2001, scientists have worked to standardize sample collection methods to validate procedures for pathogen recovery and to understand how collection methods affect pathogen detection limits. Identifying conditions that improve pathogen detection is extremely important for rapid and accurate sample recovery, analysis, and mitigation.

The Lab researchers used two methods, swabs and wipes, to collect both nonvirulent and virulent strains of B. anthracis and Y. pestis from four types of nonporous surfaces: two hydrophilic surfaces (stainless steel and glass) and two hydrophobic surfaces (vinyl and plastic). They measured sample recovery by using a real-time assay for intact DNA signatures from the pathogens. The quantitative polymerase chain reaction (qPCR) assay fills a bioforensics gap in cases where bacteria on a surface are no longer viable, or are viable but cannot be cultured.

The scientists found that collection efficiency was more surface-dependent for virulent strains than nonvirulent strains. For the two nonvirulent strains, collection efficiency was similar for all four surfaces. In contrast, recovery of virulent B. anthracis spores and Y. pestis strains from the hydrophilic glass or stainless steel surfaces was more efficient compared to collection from the hydrophobic vinyl and plastic surfaces. Therefore, collection of pathogenic B. anthracis and Y. pestis from more hydrophilic surfaces appears to increase the likelihood of DNA-based detection. These results suggest that surface hydrophobicity may play a role in the strength of pathogen adhesion. The pathogen adhesion to different surfaces is the sum of interactions between the pathogen cell membrane and the attachment surface. The surface-dependent collection efficiencies observed with the virulent strains may arise from strain-specific expression of capsular material or other cell-surface receptors that alter cell adhesion to specific surfaces. These findings aid in validating standard bioforensics procedures, and they emphasize the importance of specific strain and surface interactions in pathogen detection.

The research has been published in Letters in Applied Microbiology.  The Department of Homeland Security, Bioforensics Program (Julianna Fessenden-Rahn, LANL program manager) supported the work.

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Sample collection of bacterial pathogens from nonporous surfaces using a wipe (left) and a swab (right).