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.

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.

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

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.

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.

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.

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....

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.

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.nih.gov/pubmed/19121685?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_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.

Also - if the Serratia that I have isolated grows on a plate with the unknown white bacteria, the Serratia seems to feed off of the unknown.

Can you say why you have come to that conclusion?

The two bacteria were growing on the same plate; when incubated further, the Serratia marcescens grows on top of the white unknown. Bear with me for a minute as I give you a run down of the protocol: a small, pea-sized amount of soil is placed into a flask with 50 ml of basal salts broth, with mandelic acid as the carbon source - the idea here being that Pseudomonads can utilize mandelic acid as a sole carbon source while most other bacteria cannot. The sample is incubated on a rotary table at 30 degrees C for 24 hours, and then checked for growth. If growth is present, the sample is centrifuged at 1500XG for 3 minutes to spin down the soil, but not the bacteria. From this, 1 ml of the broth is removed and placed in a flask of fresh basal salts/mandelic acid broth, and incubated under the same conditions as the primary culture. From the secondary broth culture, plates are streaked (plates consist of the same formulation of the broth, with the addition of agar.) From this plate, the idea is that the students will get discrete colonies, and they do biochemical tests to find out if they have in fact isolated a Pseudomonas species.

On the first plate (basal salts media with mandelic acid) that I streaked with the secondary broth culture, I incubated at room temperature for 24 hours, but the growth was very sparse, so I incubated it for 2 additional days. The plate had a white bacteria, (which I'd hoped was Pseudomonas fluorescens,) but there was a single red colony growing in the middle of all of the white colonies. I took a loop and selected the single red colony, as well as a single white colony, and streaked them onto the Pseudomonas isolation agar. The following day, I looked at the initial plate, and noticed that several more red colonies appeared. In the days to follow, the red bacteria grew over the top of the red until the only thing on the plate was the Serratia. Because the Serratia grew on its own when subcultured, as did the white, I didn't think they could be the same thing. Last week, I streaked a TSA plate with one line of the red (Serratia) and one line of the white unknown. Today when I got in, the Serratia had grown over the white unknown. Even stranger, when the two bacteria are grown independently of one another, the Serratia has a waxy look to it, as does the white unknown, but when grown on the same plate, where the Serratia grows on top of the unknown, the Serratia develops a mucous-like appearance, and appears very wet and shiny.

Thanks again for any insight you can provide. Also, can you recommend a good book that would have the most up-to-date info about microbiological taxonomy?

Thanks -very interesting observation. Still think red is Serratia, but there is a Roseobacter - a Gram neg bug that can produce a little color (carotenoids), grows on organic acids and requires added thiamine and I think nicotinic acid for growth - maybe these came from the "white colony." See if it ferments glucose.

I'd just go with Bergey's. Since many species of Pseudomonas have been reclassified over the last decades to other genera what about Stenotrophomonas, Burkholderia etc or were you thinkiing Pseudomonads in a general sense? In any case. I think there aren't alot of Pseudomonas spp. that assimilate mandelic acid - aeruginosa putida. Think B. cepacia is a better chance.
My suggestion for the students id process is - streak for isolation, from an isolated colony prepare a master culture from which perform Gram stain (Gram neg rods - coccobacilli), motility (most are +), catalase and oxidase (most are cat+/ox +), glucose ferm (use TSI - note both failure to ferment and absence of growth in butt - pseud's are aerobic). Recall Acintobacters also assimilate mandelic acid but are ox -
and nonmotile.
This would give you a presumptive Pseudomonas spp. but possibly other genera so you might tell the students what to do if it doesn't key out per these tests. Pigment production on Pseudomonas agar's might help as aeruginosa of the Pseudomonas spp. is the most likely one to utlize mandelic acid but your description doesn't work so well for that bug. I'd not have the students go beyond that - other than to offer what they'd do next in identification (make them look up the keys and make rational proposal).

Good luck - hope the students enjoy the expeirment.

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.

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.

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.

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.

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?

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.

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...

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.

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

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

I use a final concentration of 20% glycerol on my frozen stocks too. I'll see about the paper in the morning; I've got a ton of stuff to do today...
Angie

Be aware that your Gram stain could be the base of the problem. Follow homebrew's suggestion. API's are pretty poor for id of environmental bugs.

due to time constraints and budget cuts, I contacted the CDC and they said they would do some tests. Their result was:
" The DNA sample yielded a 1490 bp good quality 16s rRNA gene sequence. The Top BLAST matches were to various entries for "Pseudomonas spp.". The first named species in the BLAST result is to: emb|AM411058.1| Pseudomonas putida partial 16S rRNA gene, strain 5zhy Identities = 1486/1488 (99.87%), Gaps = 0/1488 (0%). It seems that your isolate is a Pseudomonas putida or undescribed Pseudomonas spp.

Thanks to everyone that made contributions to the original question!

You are welcome.

Frankly speaking, 16s rRNA doesn't give much information and most of the time is misleading for my case. IF they can perform a flagellin typing and/with type 3 secretion system, the result will be more convincing.

Just hope that they are not trying to hide something there due to security reason.