33 replies to this topic

[Biochick99]

I attempted to isolate a Pseudomonas strain from a soil sample, and I'm not sure what I have isolated. I was able to isolate two strains of whatever it is and if anyone can provide some insight, I sure would appreciate it!

Both are resistant to Irgasan, and both can utilize mandelic acid as a sole carbon source; they are both highly motile, and gram negative. One produces a white colony on solid media and the other produces a red/fuchsia colony. The white strain will not grow at 37 degrees C, but grows fairly quickly at room temp. It stays white regardless of light exposure. The red colony grows super fast at 37 degrees C, and starts out white, but if there is any light exposure to the plate, the colonies turn red. On the mixed culture plate (from where I isolated the red and the white from,) the culture is thick shiny and mucoid. (Keep in mind that this initial plate is about 10 days old at this point.) Newer colonies do not appear as shiny or as mucoid. Both the red and white cells are extremely tiny and difficult to see, even at 100X under oil. As far as I can tell, the red culture appears to be coccoid and the white culture appears to be either a combination of very short rods and cocci, or just a lot of short rods - as I said before, they are really tiny.

Does anyone have any ideas of what this could be? I thought it was Serratia marcescens at first, but S. marcescens doesn't produce the red pigmentation at 37 C; plus it needs constant light to develop its color, and the red of Serratia is more of an orange red. My unknown red is more of a hot pink. Can anyone recommend any web sites or image galleries? Or, does anyone have any suggestions as to any biochemical tests I should do? I just did a carbohydrate fermentation test this afternoon, and I will go in and look at the results tomorrow. Please help!

[Biochick99]

The glucose and sucrose fermentation tests produced acid and gas on both unknowns, acid only in S. marcescens, and negative results in P. aeruginosa. The lactose fermentation test was negative for S. marcescens and P. aeruginosa, and had both acid and gas in both unknowns.

Biochick99, on Sep 4 2009, 09:23 PM, said:

I attempted to isolate a Pseudomonas strain from a soil sample, and I'm not sure what I have isolated. I was able to isolate two strains of whatever it is and if anyone can provide some insight, I sure would appreciate it!

Both are resistant to Irgasan, and both can utilize mandelic acid as a sole carbon source; they are both highly motile, and gram negative. One produces a white colony on solid media and the other produces a red/fuchsia colony. The white strain will not grow at 37 degrees C, but grows fairly quickly at room temp. It stays white regardless of light exposure. The red colony grows super fast at 37 degrees C, and starts out white, but if there is any light exposure to the plate, the colonies turn red. On the mixed culture plate (from where I isolated the red and the white from,) the culture is thick shiny and mucoid. (Keep in mind that this initial plate is about 10 days old at this point.) Newer colonies do not appear as shiny or as mucoid. Both the red and white cells are extremely tiny and difficult to see, even at 100X under oil. As far as I can tell, the red culture appears to be coccoid and the white culture appears to be either a combination of very short rods and cocci, or just a lot of short rods - as I said before, they are really tiny.

Does anyone have any ideas of what this could be? I thought it was Serratia marcescens at first, but S. marcescens doesn't produce the red pigmentation at 37 C; plus it needs constant light to develop its color, and the red of Serratia is more of an orange red. My unknown red is more of a hot pink. Can anyone recommend any web sites or image galleries? Or, does anyone have any suggestions as to any biochemical tests I should do? I just did a carbohydrate fermentation test this afternoon, and I will go in and look at the results tomorrow. Please help!

[HomeBrew]

You could PCR amplify the 16s gene using universal bacterial primers and have the amplicon sequenced to identify the species.

[Biochick99]

HomeBrew, on Sep 5 2009, 08:01 PM, said:

You could PCR amplify the 16s gene using universal bacterial primers and have the amplicon sequenced to identify the species.

Thanks, HomeBrew! I'll have to see if I can find some primers, but I will probably need to order some. I was also going to do some more biochemical tests this week.

[eberthella]

Serratia marcescens does produce prodigiosin at 37C. Reportedly less/not at 39C.

[eberthella]

eberthella, on Sep 8 2009, 01:18 AM, said:

Serratia marcescens does produce prodigiosin at 37C. Reportedly less/not at 39C.


Oh, and it is resistant to TCS, whatever is reported by Ciba. Curious - why did you pursue that criterion?

[Adrian K]

HI Biochick99,

Try to do oxidase test. if is oxidase positive, gram -ve, bipolar rod after staining....it probably is burkholderia (pseudomallei, arabinose -ve; thailandensis, arabinose +ve) species which i am working on.
Try use API 20NE if you do have it.

Where are you from?

[eberthella]

Red pigment? Burkholderia is unlikely and pseudomallei very unlikely and hopefully not. Esp in the western world, cepacia/cenocepaca are much or likely (sometimes a yellowish pigment). pseudomallei is a designated bioterror organism, possession of which in US can be legally problematic.

Edited by eberthella, 17 September 2009 - 03:41 AM.

[Adrian K]

eberthella, on Sep 17 2009, 07:39 PM, said:

Red pigment? Burkholderia is unlikely and pseudomallei very unlikely and hopefully not. Esp in the western world, cepacia/cenocepaca are much or likely (sometimes a yellowish pigment). pseudomallei is a designated bioterror organism, possession of which in US can be legally problematic.

True indeed. Hopefully is not B.pseudomallei but if just in case it is, please feel free to contact me.
BTW, I do have lots of pseudomallei clinical isolates. Luckily I am not in anywhere near US.

That's the reason I wonder where Biochick99 from. If he/she is from endemic region, she might probably had isolated it.

It also could be also chromobacterium violaceum which is fuchsia in colour ..and there is even non-pigmented (white) chromobacterium exist.
refer:
http://jcm.asm.org/c.../full/37/6/2068

[Biochick99]

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.

[GeorgeWolff]

It may be tough to call the species in the genus Burholderia but enterics and the Burholderiaceae are very distinct and there should no problem in their differentiation - this is micro 101. Please study these bacteria - to your isolate, pigment, Gram stain and fermentation of glucose would be all you should need. You could also toss in lactose fermentation and oxidase reaction.
I really wonder why you used some of those media - mannitol salt and esculin for example are not pivotal for identification of either and running a huge battery of sugars is not so useful.

Casual approach to id such as this usually means nothing other a person inexperienced in identification who is left to deal with the error. With B. pseudomallei, a class B bioterror agent, it's a different risk. I think that, by the US Homeland Security Act, failure to report in a timely manner possession of this bug may incur personal criminal penalities.

Edited by GeorgeWolff, 19 September 2009 - 04:54 AM.

[HomeBrew]

Move away from the phenotypic tests and think genotypic -- getting the 16s sequence is a PCR, gel purification, and a sequencing run away, and is usually pretty definitive....

[Biochick99]

GeorgeWolff, on Sep 19 2009, 04:47 AM, said:

It may be tough to call the species in the genus Burholderia but enterics and the Burholderiaceae are very distinct and there should no problem in their differentiation - this is micro 101. Please study these bacteria - to your isolate, pigment, Gram stain and fermentation of glucose would be all you should need. You could also toss in lactose fermentation and oxidase reaction.
I really wonder why you used some of those media - mannitol salt and esculin for example are not pivotal for identification of either and running a huge battery of sugars is not so useful.

Casual approach to id such as this usually means nothing other a person inexperienced in identification who is left to deal with the error. With B. pseudomallei, a class B bioterror agent, it's a different risk. I think that, by the US Homeland Security Act, failure to report in a timely manner possession of this bug may incur personal criminal penalities.

The gram stain only told me that it was a gram negative rod; there is no pigment, and the fact that it ferments glucose didn't narrow my choices that much, nor did the fact that it doesn't ferment lactose. I decided to look at all of the sugar metabolism because in Bergey's manual it is one of the characteristics that is used to differentiate between different species within the genus. My "casual approach," as you put it, is just that. I am troubleshooting a lab exercise that the students do in their micro lab where they attempt to isolate a Pseudomonas species from a soil sample. The students were getting a lot of fungi in their trials, so I decided to try it myself. I tweaked the media, and ended up getting two different bacteria in my isolation attempt. As I said before, the red bacteria was Serratia marcescens, now the white bacteria is what I'm trying to identify. I did all of the other tests (mannitol salt, MacConkey, etc.) because I had them available, and decided to use them for the hell of it. Before I call the CDC and the dept of Homeland Security, I think it would be wiser to identify what I have isolated first. I am going to ignore the jab about me being inexperienced and take the advice of HomeBrew and run a PCR on Tuesday.

[GeorgeWolff]

Sorry if you saw it as a jab - I was offering accurate comments.
Be aware that pseudomallei in addition to legal peril does offer risk of serious disease. You'd prob. not expect to find it in the western world. In a teaching context, it's problematic that you're unaware that the Enterobacteriaceae ferment glucose and do not produce oxidase whereas the Pseudomonads in general and Burkholderia spp. specifically do not ferment glucose and do produce oxidase. These are the pivotal tests you should consider at this point.
API is not real reliable for the pseudomallei
(http://www.ncbi.nlm...._DefaultReportP
anel.Pubmed_RVDocSum).
So Homebrew's suggestion might be best for you but you'll need a better idea what it is . Another approach might be t use Accugenix - but that too is not cheap.

Mannital salt agar is useful in differentating among the staph (Gram positive) and has little if any value beyond that. Irgasan (triclosan) is used in the relatively obscure Pseudomonas Isolation Agar. I understand why you might use it but be aware it's not validated for environmental bugs and has been questioned even in its proposed clinical application.

It's Burkholderia pseudomallei not "Pseudomonas pseudomallei" - that has been obsolete for over a decade. There is no such epithet tas "Providencia pseudomallei" - in fact that confuses the enteric (Providencia) with the putative pseudomallei pseudomonad. Where did you get that?

The above is largely in Bergey's.

Please - be careful with the bug and, if you're teach others, become more familiar with microbial taxonomy and the identification keys.

[Biochick99]

GeorgeWolff, on Sep 20 2009, 05:09 PM, said:

Sorry if you saw it as a jab - I was offering accurate comments.
Be aware that pseudomallei in addition to legal peril does offer risk of serious disease. You'd prob. not expect to find it in the western world. In a teaching context, it's problematic that you're unaware that the Enterobacteriaceae ferment glucose and do not produce oxidase whereas the Pseudomonads in general and Burkholderia spp. specifically do not ferment glucose and do produce oxidase. These are the pivotal tests you should consider at this point.
API is not real reliable for the pseudomallei
(http://www.ncbi.nlm...._DefaultReportP
anel.Pubmed_RVDocSum).
So Homebrew's suggestion might be best for you but you'll need a better idea what it is . Another approach might be t use Accugenix - but that too is not cheap.

Mannital salt agar is useful in differentating among the staph (Gram positive) and has little if any value beyond that. Irgasan (triclosan) is used in the relatively obscure Pseudomonas Isolation Agar. I understand why you might use it but be aware it's not validated for environmental bugs and has been questioned even in its proposed clinical application.

It's Burkholderia pseudomallei not "Pseudomonas pseudomallei" - that has been obsolete for over a decade. There is no such epithet tas "Providencia pseudomallei" - in fact that confuses the enteric (Providencia) with the putative pseudomallei pseudomonad. Where did you get that?

The above is largely in Bergey's.

Please - be careful with the bug and, if you're teach others, become more familiar with microbial taxonomy and the identification keys.

I got the Providencia from the website www.microbeid.com, which helps identify "unknowns" based on much of the same tests that are used to characterize bacteria in Bergey's. The name really confused me too, especially when I couldn't find it in Bergey's. When I typed it in on Google, it pulled up Burkholderia pseudomallei, and there were several alternate names, one of which was Pseudomonas pseudomallei - which was in Bergey's. I will admit that I do not have a whole lot of practice at identifying unknown bacteria, but I wanted to find some simple biochemical tests that the students could use to identify their unknowns. Normally, they use a nitrate reduction test and a litmus milk test, and that's it. I feel like they need to be exposed to more than just those two, especially since a lot of the students have difficulty interpreting the litmus milk results. When I did the initial glucose fermentation, it produced acid and gas; alongside the glucose fermentation, I went ahead and did a sucrose and a lactose fermentation test - both of which were positive for both unknowns. I must have done something wrong, because that totally skewed my search. So I did the OF tests to double check my results, where I found that neither strain fermented lactose, and that the red unknown (Serratia) metabolized sucrose but the white did not.
Also - why does the Serratia grow on the Mannitol salt agar? The Pseudomonas isolation agar was used only to help differentiate between the different strains of Pseudomonas, which was what I hoped I had. When the Serratia grew on it so well, that threw me off because I was under the impression that irgasan was a broad-spectrum microbial that had no effect on Pseudomonads. Do you think I should see if the bug grows on Cetrimide agar while I do the oxidase test? The reason being, if this is Burkholderia pseudomallei, I understand exactly how dangerous it could be. That's all I need is a bunch of students isolating a potential bioterrorism agent!
I do appreciate your help with this - thank you.