Impact of processing method on recovery of bacteria from wipes used in biological surface sampling

Abstract

Environmental sampling for microbiological contaminants is a key component of hygiene monitoring and risk characterization practices utilized across diverse fields of application. However, confidence in surface sampling results, both in the field and in controlled laboratory studies, has been undermined by large variation in sampling performance results. Sources of variation include controlled parameters, such as sampling materials and processing methods, which often differ among studies, as well as random and systematic errors; however, the relative contributions of these factors remain unclear. The objective of this study was to determine the relative impacts of sample processing methods, including extraction solution and physical dissociation method (vortexing and sonication), on recovery of Gram-positive (Bacillus cereus) and Gram-negative (Burkholderia thailandensis and Escherichia coli) bacteria from directly inoculated wipes. This work showed that target organism had the largest impact on extraction efficiency and recovery precision, as measured by traditional colony counts. The physical dissociation method (PDM) had negligible impact, while the effect of the extraction solution was organism dependent. Overall, however, extraction of organisms from wipes using phosphate-buffered saline with 0.04% Tween 80 (PBST) resulted in the highest mean recovery across all three organisms. The results from this study contribute to a better understanding of the factors that influence sampling performance, which is critical to the development of efficient and reliable sampling methodologies relevant to public health and biodefense.

Fig 1

Main effects plots showing the mean value of the percentages of recovery from extracted wipes as a function of the experimental factors evaluated in the study for all three organisms (A), E. coli (B), Bacillus cereus (C), and Burkholderia thailandensis (D). PDM, physical dissociation method; solution, extraction solution. Dashed lines represent grand mean. Numbers above experimental factors are P values from one-way analysis of variance. P < 0.05 was considered statistically significant.

Fig 2

Rank analysis for physical dissociation method (PDM) (A) and extraction solution (B). Block plots show mean percentage of recovery averaged across all three organisms for each extraction method. TV, touch vortexing; S1, sonication for 1 min; S2, sonication for 2 min; S5, sonication for 5 min; V2, vortexing for 2 min; V10, vortexing for 10 min; S1V1, sonication and then vortexing for 1 min each; S2V2, sonication and then vortexing for 2 min each; BB, Butterfield's buffer; BBT, Butterfield's buffer with 0.04% Tween 80; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline with 0.04% Tween 80; MRD, maximum recovery diluent.

Fig 3

Viability as a function of extraction method. A calibration curve (A) was constructed and used to calculate the percentage of recovered cells that were viable after sample processing (B). Error bars represent the standard deviation of the mean (n = 3). BB, Butterfield's buffer; BBT, Butterfield's buffer with 0.04% Tween 80; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline with 0.04% Tween 80; MRD, maximum recovery diluent. TV, touch vortexing for 10 s; S5, sonication for 5 min; V2, vortexing for 2 min.