CASE CLOSED … what really happened in the 2001 anthrax attacks?

* How did the percent of morphs in RMR-1029 compare to the percent in the Leahy sample? And if there was a discrepancy, what would explain it?

Posted by DXer on March 21, 2011

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5 Responses to “* How did the percent of morphs in RMR-1029 compare to the percent in the Leahy sample? And if there was a discrepancy, what would explain it?”

  1. DXer said

    In 1999, Dr. Worsham explained to the NAS panel:

    “So in my experience what causes the in vitro selection of oligosporogenic strains? In my hands, it was repeated passages of overgrown liquid cultures. So if you have a culture, and the organism is running out of nutrients, and the majority of the population begins to sporulate, they stop dividing. If you have a mutant in the population, that is not getting the signal to sporulate, and continues to replicate, it forms a larger percentage at the end of that culture, than it started. And if you continue to do this, it will eventually take over the culture. If you pick from the edge of colonies on a plate – particularly an old plate – that is, if you have a colony and that colony has reached a state where it would ordinarily start sporulating, sometimes what you’ll see are fingers coming out from the side of the colony, and these are mutant that have popped up, that are continuing to replicate under conditions where the parent has stopped growing and started to sporulate – and if you prepare stocks from old cultures, plate or broth, you tend to have this sort of an issue.”

  2. DXer said

    By way of some background (and people on these technical issues should be relying on documents and/or experts), the NAS report explained at page 108:

    A photograph shown to the committee of a dense lawn of B. anthracis grown on such a
    plate at Dugway reveals the presence of many papillae, small outgrowths of bacteria indicative
    of mutants that are overgrowing their neighbors in the lawn or have some other distinctive
    feature (Martin, 2010). The scientist who repeatedly prepared these materials for the multiple
    production runs told the committee that the presence of numerous papillae was the typical
    outcome. The committee believes that the presence of these papillae can be taken as evidence
    that the agar slants already contained mutants, that growth of the bacterial population on the
    blood agar plates selected for mutants, or both. Because B. anthracis cells sporulate on blood
    agar once they reach high density, the papillae could have been outgrowths of sporulationdefective
    mutants that continued to grow for several generations after other nonmutant cells
    stopped growing and formed spores.

    Second, according to one DPG scientist, the biological material (lawns, including
    papillae) scraped from these plates was then used directly to inoculate the fermentors at Dugway
    (Martin, 2010). This material was collected from the plates after the lawn population had largely
    converted to spores. Because spores must germinate before growth can resume, unsporulated
    cells (including from sporulation-defective mutants in the inoculum) would likely have had a
    growth advantage in the fermentor. Because material prepared at Dugway comprised the bulk of
    the material that was pooled in the RMR-1029 flask, and because the inocula used to prepare the
    spores had visible evidence of mutants that may have been defective in spore formation, the
    committee suggests that at least some of the morphotypes identified in RMR-1029 originated
    from Dugway. Various biological factors would have affected the resulting presence and
    abundance of the genetic variants, including their growth rates, germination rates, and
    sporulation efficiencies under the specific cultivation conditions used as well as the rate at which
    each variant arises by mutation.

    • DXer said

      At page 120, the NAS explains:

      Finding 6.3: Some of the mutations identified in the spores of the attack letters and
      detected in RMR-1029 might have arisen by parallel evolution rather than by derivation
      from RMR-1029. This possible explanation of genetic similarity between spores in the
      letters and in RMR-1029 was not rigorously explored during the course of the
      investigation, further complicating the interpretation of the apparent association between
      the B. anthracis genotypes discovered in the attack letters and those found in RMR-1029.

      Another challenge with determining the cause of the apparent association between some
      of the B. anthracis genotypes in the attack letters and those found in RMR-1029 stems from the
      possibility that the same mutations might have arisen repeatedly in other Ames strain
      populations. Colony variants with similar or even identical mutations might arise repeatedly in
      other populations for two reasons. First, we do not know the number and rate of possible
      mutations that could produce similar phenotypes. Research by Worsham and colleagues (see, for
      example, Worsham and Sowers, 1999) identified numerous oligosporogenous variants with
      phenotypes similar to those described in the letters. In response to questions raised by the
      committee, the FBI indicated that among the 296 Ames submissions to the FBIR by Worsham,
      only the Morph D genotype was detected, and it was in three samples. The A1, A3 and E
      mutations were not detected in any of the Worsham samples submitted to the FBIR (FBI, 2010a).
      Second, under the conditions used to grow B. anthracis for the large-scale production of spores,
      there may well have been inadvertent selection that favored oligosporogenic mutants, which
      would cause the frequency of these mutants to be higher than expected for mutations that
      conferred no advantage to the cells, thereby increasing the likelihood of parallel evolution in
      replicate spore productions. The recent published work by Sastella and colleagues (2010)
      highlights the possible role of repeated passage of B. anthracis in the enrichment of sporulation deficient
      mutants.

    • DXer said

      At page 88-89, the NAS explains:

      It is well known in laboratories that study bacterial spore formation (sporulation) that
      low-frequency sporulating (oligosporogenous) variants appear in a bacterial population at a
      readily observable frequency (Velicer et al., 1998; Michel et al., 1968). Furthermore, laboratory
      selective pressures can result in these variants becoming dominant members of the population
      and supplanting the sporulation-proficient parent strain. Two phenomena may increase the
      abundance of asporogenous (nonsporulating) or oligosporogenous (sporulating with reduced
      frequency) variants above that of other types of variants. When a population encounters a decline
      in nutrients, sporulation-proficient cells cease growth and enter the dormant spore state.
      Sporulation-deficient variants may continue growth to some degree at the expense of their
      neighbors, increasing their own relative abundance in the population. If this aged population of
      cells and spores is eventually transferred to fresh medium, the nonsporulated cells may also
      resume growth more rapidly than the spores, again increasing their abundance in the resulting
      population. These may be selective factors leading to the frequent observation of
      oligosporogenous variants in laboratory cultures.

      Studies of B. anthracis phenotypic variants, many of which were shown to be
      oligosporogenous, were previously carried out by Patricia Worsham and Michele Sowers at
      USAMRIID (Worsham and Sowers, 1999) as part of an effort to identify useful strains for
      vaccine research. Working with multiple strains (e.g., Ames, Sterne, Vollum, and others) of B.
      anthracis, Worsham and Sowers identified numerous phenotypic variants (Worsham and
      Sowers, 1999). Their initial screen for phenotypic variation was growth on medium containing a
      dye called Congo red. The dye accumulated by wild-type B. anthracis colonies results in the
      colonies assuming a salmon-pink color. Poorly sporulating colonies do not accumulate the dye,
      resulting in a white appearance that can be easily identified by eye. Further study of the white
      colony morphotypes revealed that they were sporulation deficient. While there is no clear
      physiological explanation for the coincidence of the absence of Congo red binding and
      sporulation deficiencies, no such explanation is required for the practical use of this morphotype
      screening method. One of these phenotypic variants was carefully characterized as carrying a
      mutation in spo0A, a primary regulatory gene for entry into sporulation (Hoch, 2000). Indeed,
      mutants of spo0A and other genes governing entry into sporulation were historically recognized
      in experiments in which cultures of cells sporulating in rich medium were maintained for
      prolonged periods of time (Michel et al., 1968).

      Reinforcing the view that laboratory conditions frequently enrich for mutants defective in
      sporulation, a recent publication by Sastalla and colleagues (2010) reports that repeated passage
      of B. anthracis on rich medium results in the accumulation of sporulation mutants, which were
      recognized by their distinctive colony phenotype. Many of the mutants contained mutations in
      the sporulation regulatory gene spo0A, as had been observed by Patricia Worsham (Worsham
      and Sowers, 1999). These included point mutations as well as deletions and insertions. Sastalla
      and colleagues further showed that the spo0A mutants exhibited a shorter lag time than did the
      wild type when inoculated on rich medium. Thus, one factor contributing to the enrichment of
      sporulation-deficient mutants upon repeated passage is that at least some sporulation mutants
      resume rapid (exponential phase) growth more quickly than does the wild type.

      • BugMaster said

        Note also that the morphs recovered from the attack material had to be able to sporulate, or were oligosporogenous.

        It seems unlikely that a non-sporulating mutant that could only exist as a vegetative cell would have survived the drying procedure, and therefore would not remain viable for long in a dried spore prep.

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