Unidentified Soil Organism - (Sep/05/2009 )

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....

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

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.

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

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.