The loss of viability with increased duration of storage provides valuable insight into the longevity of anthrax spores. Much has been written about the ability of anthrax spores to remain viable for decades, but there are few reliable data. Wilson and Russell reported that anthrax spores survived in dry soil for 60 years (16
). Jacotot and Virat found that anthrax spores prepared by Pasteur in 1888 were viable 68 years later (8
). In 1992, Bowen and Turnbull found B. anthracis
in the plaster and lagging of London's Kings Cross railway station roof space and attributed this to the use of contaminated horse hair to bind the plaster when the building was constructed a century earlier (P. C. B. Turnbull, unpublished data and personal communication). The longest claim is that of De Vos, who recovered anthrax spores from bones recovered during an archaeological excavation at a site in Kruger National Park in South Africa, which were estimated by carbon dating to be 200 ± 50 years old (4
). Conversely, Turnbull reported the loss of viability of B. anthracis
and other Bacillus
species stored on agar slants in his collection at the Centre for Applied Microbiology and Research, Porton Down, England, which occurred particularly when the lids of the bottles containing the storage slants had become loose, allowing the agar to dry (Turnbull, personal communication), a situation similar to the situation described in this study. While it is not known why this difference in viability exists for long-term storage of B. anthracis
, some factors that may play a role include the original storage conditions (dry storage versus overlay in mineral oil), drastic changes in storage (e.g., drying out of agar), or the sporulation conditions.
In this study, 85 isolates were found to harbor only pXO2. Environmental isolates of B. anthracis
lacking pXO2 have been recovered from sites with a history of anthrax spore contamination in the distant past (15
). Less frequently, isolates lacking both plasmids have been found, but, in interesting contrast to the results presented in this paper, naturally occurring isolates harboring only pXO2 have never been found in the environment. For the isolates in this study, there was no significant difference in the proportion of strains containing only pXO1 or only pXO2 over time (P
= 0.25). However, the chi-square statistic indicated that the loss of the two plasmids was interdependent (P
= 0.004). Due to the nature of the study presented here, in which all isolates were recovered and analyzed for plasmid loss at one time, it is difficult to determine the dependence of the two plasmids on plasmid stability. Ideally, in order to determine the interdependence of plasmid stability, isolates should be monitored for plasmid loss at periodic intervals over time. Thus, any conclusions based on the data provided in this study regarding interdependence of plasmid stability or loss would be speculative at best. However, Bowen and Quinn observed that some control elements governing the maintenance of both plasmids were at least partly located on pXO2 (2
Under appropriate environmental conditions, such as within sewage or in the harsh conditions of Etosha National Park in Africa, B. anthracis
could be spontaneously cured of one or both virulence plasmids (15
). As mentioned above, the collection of isolates included in this study was stored in various locations, and, although the exact history of the collection's storage is unknown, anecdotal reports indicate that the cultures were transported to various rooms spacious enough to hold a collection of this size. The inconsistent conditions of the various storage rooms in which the cultures were held may have contributed to the plasmid instability.
To our knowledge, there have been no other studies regarding the effects that different storage methods have on B. anthracis
vegetative cells or spores. However, various studies have evaluated different preservation methods. While not necessarily applicable to B. anthracis
, these studies, which mostly compared the effects of lyophilization to the effects of freezing or cryopreservation, provide some insight into the effects of preservations methods on certain bacteria. Ashwood-Smith (1
) and Sinskey and Silverman (14
) reported that lyophilization may cause mutations in DNA and/or damage to the cell membrane, resulting in increased permeability during the preservation process. On the other hand, Breese and Sharp (3
) and Sidyakina et al. (12
) concluded that while lyophilization reduced viability, it did not affect the genetic stability of Escherichia coli
. Niermans and Feldblyum reported that preservation of Bacillus subtilis
vegetative cells by lyophilization resulted in reduced viability and retention of the pTL12 plasmid in viable cells after 10 months of storage at 4°C, although lyophilization of B. subtilis
spores had no effect on viability or plasmid retention (10
). However, none of the studies covered storage periods of more than 2 years or evaluated the effect of long-term preservation on a large number of strains of any one species. In general, most reports indicated that cryopreservation is likely the best method for preserving the viability and genetic stability of strains (13
). Currently, the permanent storage method for the isolates in this study is to prepare spore suspensions from fresh cultures and to store these suspensions at −70°C in water containing 25% glycerol.
The mechanisms of plasmid loss from the B. anthracis
isolates used in this study are not known. However, it is possible that there may be some genetic damage over time that may affect plasmid replication or partitioning and result in plasmid loss upon germination and vegetative growth. It is also possible that spores stored on media (such as slants or stabs) exhibit low levels of sporulation and germination resulting from leftover nutrients from the media and amino acids and sugars released during mother cell lysis. Under these stressful conditions, nonessential elements, such as plasmids, could be cured. Stresses such as elevated temperatures (42°C) and the addition of novobiocin to laboratory media have been shown to result in the loss of pXO1 and pXO2, respectively (5
). Green et al. observed spontaneous loss of pXO2 from colonies of the Pasteur strain during growth on agar for several days under conditions conducive to capsule formation (6
). In addition, as mentioned above, Sinskey and Silverman reported that lyophilization may damage the cell membrane, causing increased permeability during the preservation process (14
). While this may not occur in B. anthracis
plasmid loss, it is another mechanism by which plasmids may be lost from the cell.
This is the first documentation of the effects of long-term storage on the plasmid stability of B. anthracis. The length of time and the storage method may have had an effect on the plasmid retention in the recovered isolates, as the majority of isolates were cured of at least one plasmid. Further studies to evaluate other media that could be used for long-term storage, as well as studies to determine the exact nature of the plasmid curing, are needed.