Unidentified Soil Organism - (Sep/05/2009 )
Good point - have these students acquired basic aspectic technique skills? Still and if Biochick is in the US or even Western Europe. pseudomallei is prob not so great a risk as it's tropical, esp. Far East.
-GeorgeWolff-
Biochick99 on Sep 19 2009, 11:17 AM said:
Okay - the red-pigmented bacteria IS Serratia marcescens; its apparently a strain that produces a lot of the red pigment.
The really scary thing is that based on the tests that I've conducted so far, the white unknown bacteria does appear to be Pseudomonas pseudomallei (aka Burkholderia pseudomallei, aka Providencia pseudomallei)- at least that's the result I get when I enter all of the info on the microbeid.com website. It is definitely gram negative, rod shaped, and motile. It doesn't grow on mannitol salt agar, nor does it hydrolyze DNA; it doesn't metabolize esculin, and it doesn't metabolize lactose. It was negative on the MR-VP tests, both at room temp and at 37C. It was negative for sulfide production, negative for nitrate reduction, negative for indole; it was slow to liqufy gelatin at room temp (it took 48 hrs) but at 37C, it rapidly liquefied gelatin. It was negative for urease but positive for citrate utilization.
I also used OF Basal medium and tested 18 different sugars, with the following results: it neither fermented nor oxidatively metabolized arabinose, cellobiose, dulcitol, lactose, maltose, rhamnose, sucrose, and xylose. It fermented all the remaining sugars, which were adonitol, fructose, galactose, glucose, glycerol, inositol, mannitol, mannose, salicin, and sorbitol.
When grown on TSA at 37C, it produced a diffusable fluorescent yellow color that is obvious under UV light, which lead me to think it was Pseudomonas fluorescens, but according to all of the tests I've done so far, its still showing up as P. pseudomallei. Also, why does this P. pseudomallei have so many different names?!?! The results on microbeid.com call it Provedencia pseudomallei, Bergey's Manual calls it Pseudomonas pseudomallei, and everyone else refers to it as Burkholderia pseudomallei.
I am trying to persuade my boss to purchase some API 20E tests, but with budget cuts, its looking kind of dim. The bacteria was isolated from a soil sample underneath a magnolia tree on the campus at Georgia Tech, (Atlanta, GA) which is where I work.
The really scary thing is that based on the tests that I've conducted so far, the white unknown bacteria does appear to be Pseudomonas pseudomallei (aka Burkholderia pseudomallei, aka Providencia pseudomallei)- at least that's the result I get when I enter all of the info on the microbeid.com website. It is definitely gram negative, rod shaped, and motile. It doesn't grow on mannitol salt agar, nor does it hydrolyze DNA; it doesn't metabolize esculin, and it doesn't metabolize lactose. It was negative on the MR-VP tests, both at room temp and at 37C. It was negative for sulfide production, negative for nitrate reduction, negative for indole; it was slow to liqufy gelatin at room temp (it took 48 hrs) but at 37C, it rapidly liquefied gelatin. It was negative for urease but positive for citrate utilization.
I also used OF Basal medium and tested 18 different sugars, with the following results: it neither fermented nor oxidatively metabolized arabinose, cellobiose, dulcitol, lactose, maltose, rhamnose, sucrose, and xylose. It fermented all the remaining sugars, which were adonitol, fructose, galactose, glucose, glycerol, inositol, mannitol, mannose, salicin, and sorbitol.
When grown on TSA at 37C, it produced a diffusable fluorescent yellow color that is obvious under UV light, which lead me to think it was Pseudomonas fluorescens, but according to all of the tests I've done so far, its still showing up as P. pseudomallei. Also, why does this P. pseudomallei have so many different names?!?! The results on microbeid.com call it Provedencia pseudomallei, Bergey's Manual calls it Pseudomonas pseudomallei, and everyone else refers to it as Burkholderia pseudomallei.
I am trying to persuade my boss to purchase some API 20E tests, but with budget cuts, its looking kind of dim. The bacteria was isolated from a soil sample underneath a magnolia tree on the campus at Georgia Tech, (Atlanta, GA) which is where I work.
API20NE sometimes will misidentify Burkholderia pseudomallei ATCC 23343 as Pseudomonas fluorescens from my labmate's experience.
Formerly identified as Pseudomonas pseudomallei, only lately it was rename as B.pseudomallei.
It could most probably be Burkholderia oklahomensis, which looks like pseudomallei but different on MLST and 16s, isolated in georgia.
reported in http://ijs.sgmjournals.org/cgi/content/full/56/9/2171
If is really Burkholderia oklahomensis ,Congratulations, you are the second on earth isolated it. I wish to have the stain if is possible.
Cheers,
Adrian
p/s: do not store any burkholderia strains in 4C refrigerator, it will cause it to die off easily. Store in room temp or do a glycerol stock culture. This is from our experiences.
-adrian kohsf-
Thanks adrian that does speak to Home brew's point. However, it's been B. pseudomallei for 16 years so that's hardly a recent change. The new epithet is problematic for Homeland Security as B. pseudomallei is specified.
-GeorgeWolff-
GeorgeWolff on Sep 23 2009, 01:44 AM said:
Thanks adrian that does speak to Home brew's point. However, it's been B. pseudomallei for 16 years so that's hardly a recent change. The new epithet is problematic for Homeland Security as B. pseudomallei is specified.
Okay, it is oxidase positive, and has not fermented the glucose in the OF media I prepared yesterday afternoon. Of course it has only been 16 hours, but still...
also, what is the optimum temp for B. pseudomallei and for P. fluorescens? My unknown grows at 37+, albeit much more slowly than it does at room temp, which is around 25C.
I sent a message to the CDC yesterday afternoon, but still haven't heard back from them.
In regards to the question about the safety of this lab - that's my argument too; if students are isolating a potentially dangerous bug, then I will have to alter the protocol so that they DON'T get this thing. Although, the protocol was in an old Micro lab manual I found.
-Biochick99-
HomeBrew on Sep 22 2009, 08:46 PM said:
Looking at this another way -- are you sure this is the best way to design a lab exercise for students? Since there are potentially harmful organisms in a natural soil sample -- as we've seen here -- and students can not be expected to possess good microbiological skills, wouldn't it be better and safer to control the experimental design more tightly?
Perhaps you can spike a sterilized soil sample with a mixture of known organisms, and have them use this soil in their lab exercise? They would get all the benefits of learning the isolation protocol and identification methods, without being exposed to potentially dangerous organisms.
Perhaps you can spike a sterilized soil sample with a mixture of known organisms, and have them use this soil in their lab exercise? They would get all the benefits of learning the isolation protocol and identification methods, without being exposed to potentially dangerous organisms.
Maybe you could consider HomeBrew's suggestion by spiking a sterilized soil for student practical.
Just curious, how deep you had dig your soil and when the time you dig it was it after raining or under a hot sunny day?
-adrian kohsf-
I basically cleared the leaves and topsoil, so the sample was taken from a depth of about an inch; it had not rained recently when the sample was taken. Since I'm in Atlanta, GA, USA, and the sample was taken in August, it was pretty hot and humid outside; if I had to guess, I would say the temp was at least 35 degrees C.
On the link that Adrian shared, the B. oklahomensis was actually isolated in soil samples in GA. So I'll have to look into that too. The TSI test was yellow in the butt, red in the slant, no gas production and no H2S production, so it does ferment glucose. I tried growing it on cetrimide agar, and it grew, but it had an odd pattern. I'll have to bring my camera with me and take a picture of it tomorrow to share.
As far as the students go, I'm not really sure if they understand the importance of aseptic technique; that's one issue I've tried to stress with the TA and with the professor. I just write the protocols, prepare the media, and basically work in the background. Of course, I have 35 different labs to prep for including genetics, cell biol, introductory labs, ecology, and even the independent project labs, so sometimes I just assume the professors and TA's will take up the slack and stress the importance of sterility. Bad assumption, huh? Regardless, I try to write each protocol with detailed instructions on asepticism and often check in on the lab and scold those that fail to follow directions. Most of them are pretty good about it.
-Biochick99-
Biochick99 on Sep 25 2009, 12:45 AM said:
I basically cleared the leaves and topsoil, so the sample was taken from a depth of about an inch; it had not rained recently when the sample was taken. Since I'm in Atlanta, GA, USA, and the sample was taken in August, it was pretty hot and humid outside; if I had to guess, I would say the temp was at least 35 degrees C.
On the link that Adrian shared, the B. oklahomensis was actually isolated in soil samples in GA. So I'll have to look into that too. The TSI test was yellow in the butt, red in the slant, no gas production and no H2S production, so it does ferment glucose. I tried growing it on cetrimide agar, and it grew, but it had an odd pattern. I'll have to bring my camera with me and take a picture of it tomorrow to share. ......
On the link that Adrian shared, the B. oklahomensis was actually isolated in soil samples in GA. So I'll have to look into that too. The TSI test was yellow in the butt, red in the slant, no gas production and no H2S production, so it does ferment glucose. I tried growing it on cetrimide agar, and it grew, but it had an odd pattern. I'll have to bring my camera with me and take a picture of it tomorrow to share. ......
Dear Biochick99,
Unfortunately I'm not somewhere near your place. Since you had isolated from the topsoil, I'm afraid that sooner or later GA will be declared endemic for B. oklahomensis. So Better be careful when playing with soil out there.
There is a paper by David DeShazer about the virulence of B. oklahomensis. From the abstract although it says it doesn't have much virulence but the article I mentioned earlier (Glass et al, 2006) saying the B.oklahomensis was clinical isolates. However I don't have access to it so I can't say much. It will be great if someone can PM it to me.
http://www3.interscience.wiley.com/journal...512547/abstract
Hope to see your findings in coming publication.
All the best.
p/s:
now bioterrorism bugs can be obtained anywhere near you....beware... -adrian kohsf-
adrian kohsf on Sep 22 2009, 05:13 PM said:
Biochick99 on Sep 19 2009, 11:17 AM said:
Okay - the red-pigmented bacteria IS Serratia marcescens; its apparently a strain that produces a lot of the red pigment.
The really scary thing is that based on the tests that I've conducted so far, the white unknown bacteria does appear to be Pseudomonas pseudomallei (aka Burkholderia pseudomallei, aka Providencia pseudomallei)- at least that's the result I get when I enter all of the info on the microbeid.com website. It is definitely gram negative, rod shaped, and motile. It doesn't grow on mannitol salt agar, nor does it hydrolyze DNA; it doesn't metabolize esculin, and it doesn't metabolize lactose. It was negative on the MR-VP tests, both at room temp and at 37C. It was negative for sulfide production, negative for nitrate reduction, negative for indole; it was slow to liqufy gelatin at room temp (it took 48 hrs) but at 37C, it rapidly liquefied gelatin. It was negative for urease but positive for citrate utilization.
I also used OF Basal medium and tested 18 different sugars, with the following results: it neither fermented nor oxidatively metabolized arabinose, cellobiose, dulcitol, lactose, maltose, rhamnose, sucrose, and xylose. It fermented all the remaining sugars, which were adonitol, fructose, galactose, glucose, glycerol, inositol, mannitol, mannose, salicin, and sorbitol.
When grown on TSA at 37C, it produced a diffusable fluorescent yellow color that is obvious under UV light, which lead me to think it was Pseudomonas fluorescens, but according to all of the tests I've done so far, its still showing up as P. pseudomallei. Also, why does this P. pseudomallei have so many different names?!?! The results on microbeid.com call it Provedencia pseudomallei, Bergey's Manual calls it Pseudomonas pseudomallei, and everyone else refers to it as Burkholderia pseudomallei.
I am trying to persuade my boss to purchase some API 20E tests, but with budget cuts, its looking kind of dim. The bacteria was isolated from a soil sample underneath a magnolia tree on the campus at Georgia Tech, (Atlanta, GA) which is where I work.
The really scary thing is that based on the tests that I've conducted so far, the white unknown bacteria does appear to be Pseudomonas pseudomallei (aka Burkholderia pseudomallei, aka Providencia pseudomallei)- at least that's the result I get when I enter all of the info on the microbeid.com website. It is definitely gram negative, rod shaped, and motile. It doesn't grow on mannitol salt agar, nor does it hydrolyze DNA; it doesn't metabolize esculin, and it doesn't metabolize lactose. It was negative on the MR-VP tests, both at room temp and at 37C. It was negative for sulfide production, negative for nitrate reduction, negative for indole; it was slow to liqufy gelatin at room temp (it took 48 hrs) but at 37C, it rapidly liquefied gelatin. It was negative for urease but positive for citrate utilization.
I also used OF Basal medium and tested 18 different sugars, with the following results: it neither fermented nor oxidatively metabolized arabinose, cellobiose, dulcitol, lactose, maltose, rhamnose, sucrose, and xylose. It fermented all the remaining sugars, which were adonitol, fructose, galactose, glucose, glycerol, inositol, mannitol, mannose, salicin, and sorbitol.
When grown on TSA at 37C, it produced a diffusable fluorescent yellow color that is obvious under UV light, which lead me to think it was Pseudomonas fluorescens, but according to all of the tests I've done so far, its still showing up as P. pseudomallei. Also, why does this P. pseudomallei have so many different names?!?! The results on microbeid.com call it Provedencia pseudomallei, Bergey's Manual calls it Pseudomonas pseudomallei, and everyone else refers to it as Burkholderia pseudomallei.
I am trying to persuade my boss to purchase some API 20E tests, but with budget cuts, its looking kind of dim. The bacteria was isolated from a soil sample underneath a magnolia tree on the campus at Georgia Tech, (Atlanta, GA) which is where I work.
API20NE sometimes will misidentify Burkholderia pseudomallei ATCC 23343 as Pseudomonas fluorescens from my labmate's experience.
Formerly identified as Pseudomonas pseudomallei, only lately it was rename as B.pseudomallei.
It could most probably be Burkholderia oklahomensis, which looks like pseudomallei but different on MLST and 16s, isolated in georgia.
reported in http://ijs.sgmjournals.org/cgi/content/full/56/9/2171
If is really Burkholderia oklahomensis ,Congratulations, you are the second on earth isolated it. I wish to have the stain if is possible.
Cheers,
Adrian
p/s: do not store any burkholderia strains in 4C refrigerator, it will cause it to die off easily. Store in room temp or do a glycerol stock culture. This is from our experiences.
Hi Adrian,
I grew up a culture last night and then diluted it in 25% glycerol and put it at -80 degrees C. If it is Burkholderia, will it survive? Thanks for the tip about the API20NE. I did find a very useful website that lists the primer sequences that should be used in determining if the culture is definitively B. pseudomallei. I'm going to try and order the primers next week.
-Biochick99-
adrian kohsf on Sep 25 2009, 04:02 AM said:
Biochick99 on Sep 25 2009, 12:45 AM said:
I basically cleared the leaves and topsoil, so the sample was taken from a depth of about an inch; it had not rained recently when the sample was taken. Since I'm in Atlanta, GA, USA, and the sample was taken in August, it was pretty hot and humid outside; if I had to guess, I would say the temp was at least 35 degrees C.
On the link that Adrian shared, the B. oklahomensis was actually isolated in soil samples in GA. So I'll have to look into that too. The TSI test was yellow in the butt, red in the slant, no gas production and no H2S production, so it does ferment glucose. I tried growing it on cetrimide agar, and it grew, but it had an odd pattern. I'll have to bring my camera with me and take a picture of it tomorrow to share. ......
On the link that Adrian shared, the B. oklahomensis was actually isolated in soil samples in GA. So I'll have to look into that too. The TSI test was yellow in the butt, red in the slant, no gas production and no H2S production, so it does ferment glucose. I tried growing it on cetrimide agar, and it grew, but it had an odd pattern. I'll have to bring my camera with me and take a picture of it tomorrow to share. ......
Dear Biochick99,
Unfortunately I'm not somewhere near your place. Since you had isolated from the topsoil, I'm afraid that sooner or later GA will be declared endemic for B. oklahomensis. So Better be careful when playing with soil out there.
There is a paper by David DeShazer about the virulence of B. oklahomensis. From the abstract although it says it doesn't have much virulence but the article I mentioned earlier (Glass et al, 2006) saying the B.oklahomensis was clinical isolates. However I don't have access to it so I can't say much. It will be great if someone can PM it to me.
http://www3.interscience.wiley.com/journal...512547/abstract
Hope to see your findings in coming publication.
All the best.
p/s:
now bioterrorism bugs can be obtained anywhere near you....beware...Adrian:
Which paper do you want - the one by DeShazer or the one by Glass? I'll see if I can get access to the one by DeShazer; I have the one by Glass. I could email it to you.
Angie
-Biochick99-
Biochick99 on Sep 26 2009, 01:05 PM said:
Adrian:
Which paper do you want - the one by DeShazer or the one by Glass? I'll see if I can get access to the one by DeShazer; I have the one by Glass. I could email it to you.
Angie
Which paper do you want - the one by DeShazer or the one by Glass? I'll see if I can get access to the one by DeShazer; I have the one by Glass. I could email it to you.
Angie
Hi Angie,I have the Glass paper. I do not have the DeShazer paper, it will be good if you can pm me.
When I do stock culture for -80 storage, I use a overnight grow 800ul volume, and add another 800ul 40% glycerol solution to make a final 20% glycerol stock culture. I guess logically if you are making 25% glycerol stock that will work as well. however I haven't try it before. But for sure never ever store in +4C fridge, B.pseudomallei will easily die off and I had a bad experience to revive most of my cultures (which my senior left for me) and lost some of my strains.
All the best.
Adrian
-adrian kohsf-