Evaluation of two surface sampling methods for detection of Erwinia herbicola on a variety of materials by culture and quantitative PCR

Abstract

This research was designed to evaluate surface sampling protocols for use with culture and quantitative PCR (QPCR) amplification assay for detection of the gram-negative bacterial biothreat simulant Erwinia herbicola on a variety of surface materials. Surfaces selected for evaluation were wood laminate, glass and computer monitor screens, metal file cabinets, plastic arena seats, nylon seat cushions, finished concrete flooring, and vinyl tile flooring. Laboratory and test chamber studies were performed to evaluate two sampling methods, a sponge and a macrofoam swab, for detection of E. herbicola on surface materials. In laboratory trials, seven materials were inoculated with a known concentration of E. herbicola cells and samples were collected from the surfaces of the materials to determine sampling efficiencies. Culture analysis was ineffective for assessing E. herbicola collection efficiency because very few culturable cells were obtained from surface samples. QPCR demonstrated that E. herbicola DNA was present in high concentrations on all of the surface samples, and sampling efficiencies ranged from 0.7 to 52.2%, depending on the sampling method and the surface material. The swab was generally more efficient than the sponge for collection of E. herbicola from surfaces. Test chamber trials were also performed in which E. herbicola was aerosolized into the chamber and allowed to settle onto test materials. Surface sampling results supported those obtained in laboratory trials. The results of this study demonstrate the capabilities of QPCR to enhance the detection and enumeration of biocontaminants on surface materials and provide information on the comparability of sampling methods.

FIG. 1.

Flow chart summarizing sponge and macrofoam swab sample processing and E. herbicola DNA extraction and purification procedures. Abbreviations: PB, 0.01 M PB; TBS, Tris-buffered saline with 0.1% Tween 20; HA, mixed cellulose esters; PBT, 0.01 M PB with 0.05% Tween 20; BSA, bovine serum albumin; TE, Tris-EDTA buffer.

FIG. 2.

Summary of QPCR results obtained from surface samples in the experimental room. Materials were contaminated by aerosolization of E. herbicola into the room, followed by settling of the bioaerosol onto surfaces in the room. Duplicate surface samples were collected by sponge (930 cm² = 1 ft²) and macrofoam swab (317 cm² = 49 in²) for each of three releases. Bar heights represent the mean of six samples ± 1 standard error.