Unidentified Soil Organism - (Sep/05/2009 )
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!
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:
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!
You could PCR amplify the 16s gene using universal bacterial primers and have the amplicon sequenced to identify the species.
HomeBrew on Sep 5 2009, 08:01 PM said:
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.
Serratia marcescens does produce prodigiosin at 37C. Reportedly less/not at 39C.
eberthella on Sep 8 2009, 01:18 AM said:
Oh, and it is resistant to TCS, whatever is reported by Ciba. Curious - why did you pursue that criterion?
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?
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.
eberthella on Sep 17 2009, 07:39 PM said:
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/cgi/content/full/37/6/2068
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.