SPADA Draft Documents

Guidelines for Verifying and Documenting the Relationships betweenMicrobial Cultures 1

1.0 Background

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Microbial cultures are dynamic systems that can accumulate inheritable changes when 3 propagated or stored in laboratory environments. These changes often affect key virulence traits 4 that are targeted during the development and testing of medical countermeasures and pathogen 5 detection assays. For example, laboratory-propagated Francisella tularensis and Coxiella 6 burnetii tend to lose distinctive surface antigens that protect them from the host immune 7 response (1, 2, 3). When cultured at 37°C, Yesinia pestis frequently jettisons the pCD plasmid, 8 which encodes a number of key virulence genes associated with the bacteria’s type II secretion 9 system (4, 5). In yet another example, laboratory-acclimated Bacillus anthracis is less likely to 10 sporulate (6). 11 These laboratory-acquired mutations have the demonstrated potential to generate conflicting 12 results in laboratories that are nominally working with the same strain. For example, 13 investigators at the United States Army Medical Research Institute of Infectious Diseases 14 (USAMRIID) found as much as a 16-fold difference in virulence of internally and externally 15 sourced F. tularensis Schu S4 cultures (David Waag, personal communications) that appears to 16 be attributed to a laboratory-acquired frame shift deletion in the known virulence determinant, 17 FTT_0615C (7). Similarly, Molins et al. (8) noted that their version of F. tularensis Schu S4 18 exhibited decreased virulence compared to other Type A strains. 19 Consequently, the research community would benefit from a consensus standard for tracking 20 provenance of microbial stocks used in different applications. This need is especially critical for 21 microbiologists involved in development of various health care applications such as diagnostics, 22 vaccines and therapeutics. Microbial reference materials used in these applications are obtained 23

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