Dr. Patricia Worsham and her research group at USAMRIID initially identified morphological variants that were either present at low numbers in the attack material, or arose during the course of cultivation of the attack material. These variants were selected solely by visual inspection of colonies that grew up on Bacillus anthracis growth medium - so this is a classical old-school microbial study, very similar to the kind of work done in, say, the 1950s.
For comparison, I would refer those interested to work such as this:
CRITERIA FOR THE IDENTIFICATION OF BACILLUS ANTHRACIS
JOSHUA M. LEISE, CHARLES H. CARTER, HAROLD FRIEDLANDER, AND
SAMUEL W. FREEID
United States Army Chemical Corps, Fort Detrick, Frederick, Maryland
Received for publication October 24, 1958
The basic point is that without the associated genetic analysis, this (rather primitive) work would never be admissible in a court of law. The curious thing about it is that there was apparently almost no communication between the Worsham group that isolated the morphs and the people who developed the genetic assays for the morphs, along the lines described by Dr. Paul Keim in his testimony.
According to an article in Nature News,
Patricia Worsham and her colleagues had noticed differences in shape, colour, and rate of spore formation even within a single anthrax culture. Ravel's team identified the genetic mutations associated with four variants and developed an assay for one of them, called Morph E. Researchers at Commonwealth Biotechnologies in Richmond, Virginia, and the Midwest Research Institute's Florida Division in Palm Bay created assays for three other variants.
Note that the Commonwealth Biotechnologies has a clear financial interest in seeing biological warfare research funding expanded (via a press release):
Since 1999, Commonwealth researchers have managed or participated in awarded biodefense grants exceeding $20,000,000 in value. CBI also operates a secure BSL-3 containment facility able to receive and work with select agents as defined by the Centers for Disease Control...
The Midwest Research Institute is a similar entity, closely linked to the Battelle Memorial Institute (via a press release):
MRI is a nonprofit research organization whose research encompasses areas such as energy, national security and life sciences. It had consolidated operating revenue of $387.8 million in fiscal 2009, which ended June 30. It has 1,800 employees across the country.
MRI also jointly manages at least one DOE lab in a joint partnership with Battelle Memorial Institute.
So, the take-home message here is that unlike the assays for the Ames variants, developed at Northern University by Paul Keim, under one roof at a public university with extensive validation and multiple refereed publications, the morph assays were developed in isolation from one another, with three of them created at secretive private institutions closely allied to Battelle Memorial Institute, one of the few institutions known to have access to both the Ames strain as well as cutting-edge spore purification and aerosolization capabilities, developed under contracts with Dugway, the CIA and the DIA. Unlike with the Ames work, there seems to be a serious lack-of-transparency issue here - one apparently glossed over by the both the committee and by Dr. Worsham.
Why didn't the FBI instead use the best-equipped and best-validated lab - the Keim lab- the one that had developed the Ames assays, all under stringent, court-admissible guidelines? Why did they farm the assays out to multiple private entitites? And why wasn't any of this brought up in the following testimony on the identification of the morphs?
All that the Chair of the NAS committee said was that "we'll get to them eventually" - but who, precisely is "them" - the complete list, that is? And when is "eventually"? Why were they not present to give supporting (or not) evidence?
The original audio that this was transcribed from is available at the NAS website:
http://www.nationalacademies.org/newsroom/nalerts/20090925.html
Slides were shown with this presentation, but as with other testimony, the NAS did not see fit to post them online. There are a lot of issues here - but one main question pops up in the very first statement: what are the FBI restrictions, and how can the NAS committee do its work under "limited release"?
Transcript begins:
Dr. Patricia Worsham: One thing I probably should mention before I start is that I am under limited release from my non-disclosure agreement which means - I just wanted to mention I'm under limited release from my non-disclosure agreement.
So, anyway, in fall of 2001 the Leahy letter was brought to the United States Army Research Institute of Infectious Diseases, to the Special Pathogens Unit, where the FBI had asked them to analyze the material. As part of that analysis, it was plated on sheep blood agar, and in one case the plates were left in the incubator a couple of extra days, and Terry Abshire? who is an excellent microbiologist and has a good eye, noticed that there was something unusual about the plates, and in discussion with other people in their division an in ours, she recalled that my lab had published a paper some years before describing certain variants that arise during laboratory culture of Bacillus anthracis.
So, this is not the same plate that Terry had, but this is very similar. This is the more typical kind of Bacillus anthracis colony - it's gray, it's pitted, you tend to find some medusa forms, but you notice these sort of creamy yellow-colored colonies here - that's not the way Bacillus anthracis typically looks. But it is very characteristic of the variants that we had described years earlier that were defective in sporulation. This is just a close-up of some of them.
So, Terry made this observation that after long-term incubation of plates - they're not nearly as obvious after 24 hours or so - the samples contained more than one colony type, and that these aberrant types were stable, that if you picked them and streaked them out for isolation they maintained that unusual phenotype. They didn't revert back. So the question is, was this a mixed culture or was this an artifact produced by the long incubation? Because in our experience it is this kind of long incubation that gives you this aberrant morph type - and if it is a mixed culture, can we use that phenomenon, that thing, to trace the origins of the sample?
So I haven't published all of this, but this is work before the FBI, so I can say whatever I want. Stable variants can be isolated from infected animals - I've seen them come out of guinea pigs, I've seen them come out of vaccinated animals, by phage selection and during laboratory culture. Not all of these are oligosporogenic or asporogenic, there are various kinds of variants you can see. The type and the frequency of the variants appears to be strain-dependent - and one thing that I had not noticed several years before this is the Ames strain is one of the strains that tends to throw off strains that are defective in sporulation - and some of the most common variants found in laboratory culture are oligosporogenic - that is, they are not asporogenic, they are capable of making spores, but they make fewer spores under conditions where Bacillus anthracis would normally make spores, or they will only make spores under certain conditions.
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.
The phenotype is that they're generally larger, a more spreading colony morphology, you lose some of that characteristic medusa formation, there's a slight cream yellow colony pigment on sheep blood agar, so they're not as grey as typical Bacillus anthracis and they are somewhat hemolytic, which is why when I first stumbled across these I was convinced that they were not Bacillus anthracis. If you put them on sheep blood agar and leave them for a couple of days, they will be hemolytic, or you can sometimes observe it in younger cultures by say, taking a 20-hour blood agar plate and putting it in the refrigerator for a week. When you come back, and this is probably the sulfhydryl-based hemolysin that anthracis has.
This reminds me that the Congo Red issue is how I initially found these variants many many years ago. And that is, I was interested in looking at virulence in surface proteins, and one of the ways - I'm from a gram-negative background - that we often look for changes in the surface properties of an organism, is by binding of dyes. And so I incorporated Congo Red into the growth media with Bacillus anthracis and I began to find that the wild-type formed pink colonies but then these sort of white outgrowths would appear - and those were actually oligosporogenic. They have decreased amounts of the surface array protein, EA1, there's a brown pigment that they make which is a byproduct of catechol metabolism, and there's diminished as I said spore production. I was interested in these from a virulence perspective, I tested the vegetative cells of some of these strains for virulence, and they really weren't impaired compared to the wild-type, and that was pretty much the end of my love story with these strains for quite a while, because they didn't seem to be important to virulence, and I had to move on.
QUESTION: In your Canadian Journal of Microbiology paper, you pinned one of these down to -inaudible- which is part of the set of genes that drive entry into sporulation.
Correct.
QUESTION: Did you characterize any other mutants or did they generally prove to be response regulators, or the relay, or components of the relay, or did you not go beyond that?
I have a large collection in storage in the freezer, which I've never gotten back to. I think it would be an interesting thing to do. I'm guessing from the phenotypes that some of them are other early events.
QUESTION: They look like early blocks...
The spoO series, a lot of them look like that, but I haven't done the genetic studies to prove that. This is pre-FBI.
So, our goal was when the FBI came and asked us to work on this, and I should mention that Sargeant First Class Melissa Hunter was a big part of this - we worked together on this project to determine if the original samples contained more than one stable variant of Ames or other strains or other organisms - to purify and characterize the variants present in the original samples and submit them for genetic analysis - and identify any recognizable phenotypes that might lead to genetic fingerprinting of variants and potential identification of source material. Part of the challenge involved with this is that the very conditions that show us the difference between wild-type and variant are the ones that encourage spontaneous development of this type of mutant, and that was a difficult thing to get around.
So, as you grow a culture long-term, comparing variant and wild-type, it becomes easier and easier to see the difference between them, but on the other hand you run the risk of selecting novel mutations along the way, because that's what promotes this kind of mutation. So, is it a variant in the original population of spores, or is it a variant produced by spontaneous mutation favored over the wild-type, under these growth conditions - and in this case, the variants, they would have been artifacts, and not present in the original sample.
So, our solution to this was to minimize the number of generations in the laboratory between germination of the evidentiary material and storage of archival samples. We performed the genetic analysis, or I should say the people who did the analysis did it on material cultured directly from the archival samples - and we stored a second set of samples, and I'll explain this a little bit more in a minute - after another round of laboratory passage, it gave us more material to work with, in terms of doing phenotypic studies.
So - the spore samples I was given among other samples to work with - powdered material from the Daschle and the Leahy and the Post -these were resuspended in sterile water, and then plated on sheep blood agar, at 37 degrees Centigrade, at 37 degrees with CO2, and at 26 degrees. And part of this was just an attempt on my part to find some way of finding the best growth conditions for distinguishing between wild-type and variant in that population, and I was kind of hunting, and that's why there's so many parameters there.
We picked colonies, and froze half of the colony directly into glycerol, and that went into the freezer as the archival stock. The other half of the colony went to a streak plate, and that's what we froze as what we call the S1 subculture one, the freezer stock. The archival stock was what was used to send sample material for genetic analysis, and the S1 was generally used for - I'll explain the pick plates in a minute - Congo Red, new sporulation medium, L-D? medium, and I'll explain this too, and gamma phage.
I described the Congo Red, that is the wild-type tends to be somewhat salmon colored and shiny, whereas the variants, if they're oligosporogenic, will be white to cream colored, flat and spreading. New sporulation medium is a solid medium, in this case a slant, and what you are looking for is production of spores, and morphology. L-D is a classic medium for growing spores, in the laboratory, and that's what we have used - it's a liquid medium, if I didn't mention that.
Genetic analysis is probably going to be talked about by the people from TIGR, gamma phage is a bacteriophage specific for Bacillus anthracis. And so it was also important to make sure that whatever we were pulling out of these samples was Bacillus anthracis before we went further with other analysis.
So as I said we took the powdered material that was diluted in sterile water, and diluted it to get between 500 and 100 colony-forming units per mL, so then when we spread a 100 microliters, we would have had between ten and fifty colonies, so that we would get sufficient separation, to be able to look at each colony clearly. The plates were incubated under various conditions, and we looked after between 18 and 22 hours for variants, and I'll show you a picture of one of those plates in a minute. We picked both typical and atypical colonies for minus 70 stocks, and the primary subcultures. We found during this process that even blood agar has variations, so the homemade blood agar that we make in our tissue culture center at USAMRIID actually worked better for differentiating at this time point than the commercial Remel? blood agar. But Remel? has its place too, as we'll see in a minute.
So this is what one of these plates looks like at about 20 hours. It's definitely not as easy to distinguish between wild-type and mutant as it was on that first set of plates that I saw, so we did a lot of squinting - as you can see down here, this one is slightly yellowish, that one is a little yellowish, so it took a lot of eye exercise to look at the sheer number of plates we went through, looking at variants in different samples.
QUESTION: What is the pigment?
The pigment? That particular yellow pigment, I don't know for sure if it's the same brown, the diffusible pigment that we see in other situations, but that particular pigment is a catechol degradation product.
QUESTION: So it's a melanin-type?
Not melanin per se, but it's more like the siderophore catechols, I think, when they're oxidized, they turn brown. That's the brown pigment I believe, because - oops - is this me? I have a bad effect on computers.
Where was I - pigment - thank you. The yellow pigment in the colony per se, I don't know if it's the same thing as the brown pigment, it may or may not be. The brown pigment is easier to visualize in liquid culture than it is in solid medium.
So this will show you, this is one of the S1 plates, half of the colony was taken from that original plating and plated on blood agar, and this would be wild-type here, and you can kind of see the medusa forms in this area - and this is one of the mutants, this is one of the variants - so you can see overall, the colonies are larger, it's hard to tell the height of the colonies from this angle, but they're more flat and spreading, and you can kind of see in this area where there is some pitting and sort of a shiny appearance, and that's very characteristic of Bacillus anthracis as it starts to sporulate, and you don't see that...
QUESTION: Are all of the morphotypes defective in sporulation to some extent? Did you actually quantify...
All of the ones that I examined for effects in sporulation were defective in sporulation, and I'll show you different degrees in a minute - there are different categories, and it's interesting.
This is just a close-up, you can see a little better the difference in the morphologies and the pitting here as this begins to go into sporulation that you don't see over here. Pick plates are something else that we invented as part of this process - we were looking for a way to differentiate between variants. It looked like we had found ways to differentiate between wild-type and variant, but how many different kinds of variants did we have? And after a number of attempts we came up with a microbiological methodology where we used fine-tipped pipet tips to pick colonies, variant and wild-type, into a pattern with control strains, so that we could compare morphology directly alongside control samples. Otherwise it's too hard to shift your eyes from plate to plate. The plates were incubated at 48 hours and 37 degrees, and then incubated for up to two weeks to assess hemolysis, and in this case we found that the Remel blood agar produces better results than the homemade, and the difference here is in the blood agar base, they're slightly different in peptone versus tryptone, you wouldn't think it would make that big of a difference, but it does.
So here's a close-up of a pick plate. Here's wild-type Bacillus anthracis down here, this is the Ames ancestor strain, which we call 1981, this is one of the morphs, this is morph A, so you can see it's much more spreading, it has a moist bull's eye appearance in the center. Another morph that we saw originally in the Leahy - again, this is wild-type down here, this one does not have the bull's eye appearance, it does not have the moist center, it tends to be more pigmented underneath. I think that this is a result of more hemolysis and that may actually be the cause of some of that yellow pigment as well, I have no proof of that, but the strains that tend to be more hemolytic also tend to have more of that yellow pigment.
This is a different type of morph, um, this is wild-type and this is a morph that is smaller, I call this one opaque originally, it forms small very dense colonies that also tend to be a little bit different in color. This is what a pick plate looks like after 48 hours. These are the ones after 48 hours. This is after it's been refrigerated. So, there is a pattern that we put on each one of these plates - this is the wild-type strain, this is what we call the A morph, the first one I showed you on that pick plate, this is the B morph, and this the C/D morph, they're really identical in terms of morphology. And these are two picks from the same colony that I'm assessing - so, these on the outside are the controls, and these are the testers. And so in this case, what we did is we picked with a toothpick or actually a fine tipped pipet tip, from a single colony, and dipped it twice into the agar, so we have two - sort of look off up here - and this is a different I should say this is wild-type it looks most like this one. In this case there is wild-type, there's wild-type, there's the A morph, the B morph, the C/D morph, and it's kind of hard to tell because the hand is behind there, but hemolysis is the same between all of these, and this is a B morph, in this case. This was also tedious.
So, just as a little summary of some of the morphs that we looked at. The colors varied from grey-white to yellow and various versions of gray and yellow. The wild-type is medusa, it binds Congo Red well, it forms compact pitted growth areas on pick plates, it is shiny and pitted on new sporulation medium, which indicates that it is sporulating, and if you look at a subculture of that you can see the spores. It makes a little bit of pigment on new sporulation medium, but not much. Sporulation on L-D is excellent at 37 degrees, and it also makes spores at 28 and I'll get to that in a minute.
The A morph, the one you saw that had the bull's eye and the sort of moist center to it, is the largest of the morphs that I identified in these samples. It is slightly hemolytic, there is no pitting on NSM, lots of pigmentation on NSM, and this is one of the ways to sort of also divide out this morph from the others. It's a poor sporulator on NSM, it's a poor sporulator on L-D, it's a poor sporulator on sheep blood agar, and the temperature doesn't make any difference.
QUESTION: So can you explain the scale? Four plus - what does that mean?
Four plus in this case would be almost entirely spores.
QUESTION: Four plus is then - five would be?
I guess it comes back - I'm trying to think of my clinical days - there was nothing bigger than four plus. I don't remember where that came from.
QUESTION: Okay, and is plus minus greater or smaller than one to two plus?
It's smaller. It means that you might ocassionally see a spore, but not all of the time.
QUESTION: So, one percent or something like that?
Yes.
QUESTION: So, two would be noticeably defective?
Noticeably defective Yeah - we were not trying to quantitate this per se...
QUESTION: I understand. So all of these morphs were defective in sporulation?
All of them were defective in sporulation. The interesting one was the opaque, which although it didn't resemble the others in the least, in terms of morphology it didn't fit all the criteria that I was looking for, I just happened to notice this small unusual organism and thought it might be something other than Bacillus anthracis - ah this one is also defective in sporulation but it's temperature sensitive. That is it will sporulate at low temperature, but not at 37. And that - that's a totally different class of morph than I had ever seen before.
QUESTION: You say that, it's only under low temperature - so what would be the degree? Is it four plus?
As I recall, it was only about three plus. It wasn't a hundred percent - but there was a definite temperature effect.
So, just as a summary, these morphs sporulate better on blood agar than then do on L-D, and the E morph, the opaque morph, sporulates more efficiently at room temperature than at 37.
QUESTION: So, organisms that are defective in sporulation, as a population goes and sporulates, you can imagine that those may go through one or a couple more divisions, right? So, as you set up sporulation preps, poor sporulators may be significantly favored.
I don't have a lot of experience with preparing spores, per se, in my background, I have done some of it - but I would say that the conditions in which I saw these things arise most often are conditions that the organism should not have been subjected to. If someone is ignoring their cultures and not being very careful about the way that they're working with them, I would say that's the kind of situation where you tend to get these things popping up.
QUESTION: So, sporulation is the result of some sort of stress - you run out of something, or you're not happy or something - so if you have a problem with it, with sporulation, you keep going.
You keep going up to a point, and then they die out.
QUESTION: So, then they die out. But if someone keeps adding media, you could imagine this population become...
Yes - you can't leave them too long or what you see is- if you put them on sporulation medium, and you leave them to the point that they're truly out of nutrients, you'll see huge long filaments, very thin filaments, just before the culture dies. So it's - I think that, under conditions where you let it go on for two days instead of one day, then you subculture it - that's a situation where you might be more likely to see these than if you let it go twelve hours.
QUESTION: And you have seen, in your experience, the same morphs come up?
I can't tell you they are the same morphs, because we haven't done any genetic analysis. Things that look the same, and that's why we even started looking at sporulation - they looked enough like it, that.
QUESTION: Did you do microscopy, to see whether these morphs were blocked early in sporulation - or use some other criteria to judge?
I would love to do that, but this was sort of a small-scale operation here - ah - there are a lot of studies that would be interesting to do. I think that would be fun.
QUESTION: That was never done?
No. I didn't do it.
QUESTION: You say they look the same. Is that just on the initial plate, or did you take any of those things that pop up and subjected them to these other tests?
Okay, let me see if I understand the question. Isolates that I had from years before?
QUESTION: Years before, or if you take your Ames ancestor strain...
And do it again now?
QUESTION: Go to it two days...
I haven't done that study. Yeah, there are a lot of interesting approaches you could use with this, but I think thats - at the time, it seemed more like a basic science interest question, that was more interesting to me than to anyone else.
QUESTION: If I follow you correctly, the fact that you recognized these morphotypes, in some ways - I don't want to put words in your mouth, but you correct me where I'm wrong - you had seen some of these things before?
Yes.
QUESTION: And they in fact looked similar to things you had seen before?
Yes.
QUESTION: Okay, that's very important, because that means some degree of reproducibility, in the ability of these things to come up.
I don't know how these things specifically arose - I can just say how I've known them to arise in the past in my hands.
QUESTION: And the ones you've seen before are from Bacillus anthracis?
Correct - and not all strains of Bacillus anthracis do this.
QUESTION: The Ames strain does?
The Ames strain does, New Hampshire does, Vollum does not, and it actually - if we can go into scientific theory here - if you look at a whole series of Bacillus anthracis strains on Congo Red, the ones that are the darkest, sort of salmon-colored, that are picking up the dye the best, are the ones which tend to be the most virulent, and also tend to throw these morphs off the most. So I think that dye uptake is indicative of something about the ability to sporulate.
QUESTION: Just going in that direction, the Ames does, and the others don't.
Some others do.
QUESTION: Some others do, some do, some don't - but the morphs that you've seen before of the Ames, just to your general approximation, are any of the morphs that are in the RMR-1029 new, or have you seen them all before?
I don't think I ever looked at any of my old morphs as much as I looked at these. I don't think there was ever the level of intensity. As I said when I first worked with those strains, I was looking for some kind of correlation with virulence, and when I didn't find it they went to sit in the freezer and they never came back out.
QUESTION: Were any of the older morphs that you have just stashed away, has anybody ever suggested doing genetic analysis on those compared to...
They were submitted to the repository and they would have been screened along with everything else.
QUESTION: So even the morphs were screened, and were any of them among the ones that came up positive for one, two or three of the genetic tests?
I don't know. I did all my work blind.
QUESTION: And the ones who know the answer to that question?
The FBI has never told me specifically whose samples came up positive.
QUESTION: By specifically, I mean the genetic identity, whether these phenotypic similarities reflect genetic identity - not whether they are the actual material that...
And then how often those particular mutations pop up.
QUESTION: Absolutely - you're very clear. [multiple voices, inaudible] of a mechanism for selection, potentially, which is this little correlation, now when we heard anecdotal evidence that these things can be recognized on a plate but we don't know whether [multiple voices, inaudible] they were a genotype and they would have turned up positive and independently...
I will say that the one thing I can sort of say to that is, um, that - and this is what Dr. Losick had referred to in terms of the earlier paper on the asporogenic strain that I isolated that is a vaccine production strain, that isolate has a deletion in spoOA and it is a rather large deletion, and so I did get excited when I saw that, and go back and screen a number of these strains to see if it had that same deletion, and none of them did. So, at least that particular one is not a hotly reoccurring event, say like the pigmentation deletion locus in Yersinia pestis which happens routinely in a specific way.
QUESTION: I was wondering how many Ames morph variant isolates would you have frozen away, would you say, from all this past work?
I'm guessing between fifty and a hundred. And I don't know that they are all unique, because they've not been analyzed, other than they were sent to the repository and presumably they've been screened in that respect.
QUESTION: So, they were sent to the repository.
We had to send every Ames strain we had to the repository, yes. It didn't matter whether it was typical Ames or not - anything that had Ames on it went to the repository.
Alright, so we did, as we went along, analyze a subset of repository samples for variants, but as you can see from the flowchart, this is a really tedious way to go about this, and you almost need the same pair of eyes to look at everything - and when you have a thousand plus samples it really seemed to be impractical to go about it that way. So certain variants were selected that had been isolated from the evidentiary material, and they were submitted for genomic analysis in order to facilitate molecular genetic assays.
So here's just some of that early work having isolated an A type and a B type along with wild type from the Leahy, the Daschle and the Post. So, basically the way that this went down - I initially screened the Leahy and I pulled out a number of variants from that, and then I went back to the Daschle and Post and tried to find similar morphotypes in those samples, and the good news is I know wild-type when I see it, and the B morphs turned out well - all three of those had the same SNP. The A morphs were a lot more complicated, in that they all have a lesion in the same locus, but they were three different lesions - so there are hot spots in the chromosome that lead to identical or seemingly identical phenotypes, but they are not genetically identical. This sort of crosses over into Jacques Ravel and David Rasko's work, so I'm not going to go into it too much, but in all three spore samples we found this is a SNP between spoOF and and open reading frame, it's probably affecting initiation of sporulation - it looks like it is an early block from what little I've done.
Opaque is interesting, it can either be a 21 base pair deletion or a 9 base pair deletion, and it is actually in PX01. This is the only morph we've found that localizes to one of the Bacillus anthracis chromosomes. And this appears to be some kind of response regulators.
QUESTION: So it could be feeding in to the phospho-relay.
It could be - and the interesting thing is that because it's on that plasmid, and so many things on that plasmid are controlled by temperature, that does make you wonder about the temperature sensitivity of this phenotype. That would be fun to pursue. The A - numerous duplication types - it's not clear what the role of this is - it's located near a 16S rRNA, an open reading frame, a polysaccharide deacetylase - maybe Jacques and David will have more input on that by now, but it's never been clear why these strains are so affected in sporulation - and the CD morphs all in the same open reading frame - since they're histidine kinase, but all different kinds of things, SNPs and indels and presumably these are acting in phosphorylation of spoOF and possibly spoOA.
QUESTION: So it looks like at least two of those have multiple different genetic basis...
Yes.
QUESTION: ...even within the same gene...
Yes.
QUESTION: ...and I'm a little confused by this paranthetical notation, "all but one unique"?
That means that there was one amorph type found in multiple samples.
QUESTION: All the others were only found in one sample?
That's the last I've heard - now, I don't know how many more they've sequenced, if they have, but there were an incredible number of A types of all different genotypes.
QUESTION: Say that one more time? That's, that's... the morph was.. so saying the genotype was only found...
So, so the phenotype...
QUESTION: The genotype of the morph when subjected to sequence analysis...was more different...
Okay, so let's define what we mean by morph. Morph is phenotype - so, same phenotype, same locus, different types of mutation. Many different kinds of mutation.
QUESTION: So, do you think that - so, when you were doing these experiments, at the beginning you sort of raised the possibility that if you had to distinguish between those that had been outgrowth on your plates where you were characterizing the phenotype versus those that you thought were present in the admixture, if there is any way to relate that to whether you're one off?
Well, but the genetic analysis was done only on material directly from that first culture plate, so it wasn't from subcultures.
QUESTION: So, spores were plated out...
Yes.
QUESTION: ...in multiple colonies...
Yes.
QUESTION: ...and things that had this A-like phenotype were picked and then separately analyzed and each one...
Yeah. Can I draw - would that help? So, you can't really say it's an A morph when it first comes out, so you have four plates - you put int on plates and you pick what looks like aberrant colonies and some wild-type to. But all you can say at this point is that that looks a little bit different than the others - it's a little bit more yellow, a bit bigger, a bit flatter. Half of this colony goes into the -70. Half of it goes onto another plate. That plate is then -[inauduble]- That is the S1. So, most of the phenotypic studies, if I did repeats for example, using this material, I would use this, but this is what was submitted for genetics. Did that make sense?
QUESTION: They then did the DNA extractions directly from that -70 stock?
No, no they didn't do it directly from that -70, it was done as a primary overnight culture.
QUESTION: An overnight culture?
Well, like, from a freezer vial to a plate, and harvested the next day.
QUESTION: How are the cultures purified? Just kind of squished on a plate and gathered up?
Right, right.
QUESTION: Any reason they put it on a plate and not just do a 5 mL overnight?
Well, I was the one who decided that, probably. I would say in general you don't want to go from a glycerol stock to liquid, or I don't. It's easier to tell what kind of growth you are getting on a plate, it's less stressful on the organism, because they're more concentrated in one place, they can modify the microenvironment, so in general I never go from a -70 to a liquid culture.
QUESTION. Alright - so then, as a test - then, if you went back, and looked at the cells on the right that got streaked out and put in the freezer, do you see the same mutation - the identical sequence?
I didn't do that, so I...
QUESTION: That was never done?
I don't know if it was done or not. I prepared a lot of samples, I don't know what was done with all of the samples, I...
QUESTION: That would be a good test!
I can only tell you what I've done because some of it was outside my purview. I - we did harvest material from these and these also but I can't tell you exactly what was done with them.
QUESTION: They still exist?
The FBI has the samples.
QUESTION: So, either the frozen culture or the -70 culture - if you restreaked, do you see any reversion -[inaudible]-
No.
QUESTION, Always, it is an -[inaudible]-
Right, the only -[inaudible]- I've seen is - nd it's not reversion, if you take something that's, and this is going to be a -[inaudible]-, that's intermediate in genotype, that's only partially defective in sporulation, and you subject it to the same miserable conditions that causes mutants to pop up, you'll push it to where it makes fewer and fewer spores. So, I think you're getting secondary mutations in that case that pushes it more towards the asporogenic type. Does that make sense? But I've not seen them revert to wild-type.
QUESTION: It may not be to wild-type, it may be from A to B.
I've never seen them go in that direction - I've seen with other samples C-type things go to A or B. That is, it's somewhat productive, but if you put it under the right conditions it can be -[inaudible]-. And I think, Jacques and David can probably answer some more of these questions.
So, what did we conclude? That more than one stable variant similar to previously characterized strains was identified. The appearance of the variants as single isolated colonies after only eighteen hours of incubation suggested that the original samples contained a mixed population, and I think that there have been other studies that the FBI has done that perpetuates that idea, and a subgroup of those variants were submitted for sequencing, if you leave the plates longer, you see additional variants, but as we said, if you incubate the plates longer it's not clear why you're seeing those additional variants.
I also did a little bit of comparison of random colony picking versus, ah, the enrichment that I've described to you where you look for variants on a plate - just to see if it was really doing any good to look for them, or whether it was just as good to go randomly. So, the protocol enriched for most variants over the wild type. So if I picked colonies that looked like variants as opposed to picking them randomly, I did much better at finding variants, the exception being the opaque variant, E, in which case I was better off picking randomly to find that one, than in spotting it, and that has something to do with the bias in the visualization of that particular mophotype. We always got some colonies that we picked that we thought looked aberrant, but which turned out not to be - I think part of this is just the physical difficulty of looking at plates in this way. Colonies on the edge of the plate always look a little funny. Colonies that are coming from spores that are probably late to germinate are smaller and harder to assess, and those in close proximity to another colony sometimes look different as well. So that's one of the limitations of this.
So we isolated stable variants from evidentiary material. It was facilitated by an enrichment scheme based on morphology, we characterized them, placed them into classes based on phenotypic traits, and in many cases those with the same classes and similar though not necessarily identical genetic lesions, and in some cases the same variant - I should say some, rather than say - in some cases, the same variant genotypes were found within more than one evidentiary sample. Questions?
And actually while I'm thinking about it, I'll answer your question about Ames. The reason that Ames was a popular strain comes back to a paper from Steve Liddle back in the early 1980s I believe showing a group of Bacillus anthracis strains and their ability to overcome certain vaccines, and Ames was one of the ones that appeared to be vaccine refractory to a certain extent - that is, you couldn't protect animals as well against Ames - and that's what led it to be a popular challenge strain for vaccine studies. The other problem with Vollum, which had been used in the past, is that there - at least in my experience - there are a lot of Vollums out there, and no two of them look alike. So Ames had a good history, it had not been passed in the laboratory a great deal, and that's why it was a popular strain. Yes?
QUESTION: I know this is pure speculation on my part, but did you say that qualitatively, the portions of the different morphs in the different samples corresponded to your qualitative grading of their sporulation propensity?
I don't have a lot of experimentation to prove that, but I will say the ones that tended to be not totally defective in sporulation, that seemed to be in that intermediate area, were more likely to be seen than the ones that were severely affected. But I don't have the statistics to back that up.
QUESTION: I have a feeling that this wasn't something you directly did, but in working, to the best of your knowledge, in working with the design of the genomic tests to screen for these variants, did they consider whether those tests would have reacted to some of the similar but not identical mutants that you found -in other words, if they're involving rearrangements and you're designing a primer across those, could you tell if it was an 8 base pair deletion, versus a 12 base pair deletion?
I believe that they will answer that - I'm sure that - they're coming to speak, correct? Jacques? Rasko? Somebody?
CHAIR: Yes - we'll get them eventually, yes.
They were - then answer to your question is as far as I know yes, they can tell the difference - but they need to answer that question themselves.
QUESTION: They knew about the different...
Oh, yes. Well, they had to because they were the ones who sequenced them, yes. They were the ones who did the original sequences so they knew of all the variability. Anything else?
QUESTION: Do you know of anyone else in anthrax science who is also working on these morphs? Or is this something that you had come across...
It was pretty obscure so I think I was the only one interested in it for a long time. There's been more interest in them from a sporulation point of view and how it reflects control of sporulation. That's the most interest that I've gotten from it. Thank You.
CHAIR: Thank you very much, I want to see if there are any further questions from anyone else. Dr Keim is still here... - [audio cut off in mid-sentence] -
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