why biochemical identification and DNA identification shows different results? - (Mar/25/2008 )
Dear Friends,
Would you please help me if you have any idea about my results:
I isolated lactic acid bacteria from marine sample using MRS agar. then I restreaked it to modified MRS agar (supplied with catalase and sodium glycerophosphate) twice to increase the purity of the isolates and I was very careful about contamination. after that I did some biochemical identification test as well as API 50 CHL kit. then I found through the API my isolated strain is lactobacillus fermentum. then to confirm this results I did genetically Identification using 16S rRNA. then after getting the DNA sequence and running BLAST I found Staphylococcus not lactobacillus!!!!!! how come???
why different results through biochemical and DNA identification from same culture? I suspected for lactobacillus but got totally different strain on same agar (MRS agar) what do you think???
Thank you.
Leonardu
Looking forward to hear suggestion.
Would you please help me if you have any idea about my results:
I isolated lactic acid bacteria from marine sample using MRS agar. then I restreaked it to modified MRS agar (supplied with catalase and sodium glycerophosphate) twice to increase the purity of the isolates and I was very careful about contamination. after that I did some biochemical identification test as well as API 50 CHL kit. then I found through the API my isolated strain is lactobacillus fermentum. then to confirm this results I did genetically Identification using 16S rRNA. then after getting the DNA sequence and running BLAST I found Staphylococcus not lactobacillus!!!!!! how come???
why different results through biochemical and DNA identification from same culture? I suspected for lactobacillus but got totally different strain on same agar (MRS agar) what do you think???
Thank you.
Leonardu
Looking forward to hear suggestion.
if you look at your bacteria with a microscope it would be easy to determine if you have lactobacillus (rods) or Staphylococcus (cocci)...I know its oldfashioned but it may save a lot of work and headache
But reguarding your problem: the MRS agar and the API 50 CHL kit are - as far as I remember - for the ID of Lactobacilli and other bacteria form milk products. Therefore the bacteria growing on this medium may show preferences for lactose, galactose or other "typical" compounds utilises by Lactobacillus or bacteria from milk(prod). So you have some kind of isolation and identification bias here. It is possible that a Staphylococcus adapted to these food sources and gives a similar profile as l. fermentum (how good was your API-ID score btw?). Working with API 20E we frequently ID Yersinia pestis
but until now it always turned out to be some kind of Enterobacter 
But as mentioned above: look at your Bacteria and if you find rods, you can start worrying...but even BLAST is not without mistakes!
good luck!
Thank you gebirgsziege, I have already checked the cells through microscope. it was short rod not cocci and my culture is catalase negative but this Staphylococcus is catalase positive. the score for API 50 CHL got 99.6 %ID. As you mentioned lactobacillus is rod in shape but I found in some references they said lactobacillus also can be find in short rod!
Have you seq the same culture -ie from the same agar plate- that you have used for API?
Are some people in your lab working with Staphylococcus...maybe your PCR/DNA extraction reagents got cross contaminated or you got some contamination into your pure culture (must not be visible...PCR bias)?
How good was your BLAST result and/or how reliable were the Seq of Staphylococcus in Genbank (ie. were the bacteria environmental samples or did you get strains from ATTC/DSMZ etc.)?
If you are sure that your bacteria are rods, and all the "old fashioned" test point in that direction I would consider it as Lactobacillus! I understand your problem, as at the moment there seems to be a trend to not look at the organisms you work anymore. Just go for a seq and label your petri dish....or people are working with contaminants (no joke, I know more than one example where people started working with one organism and after a few weeks/month/years found out that they have been investigating something completely different
)g
These isolates are dominated isolates in my MRS agar plates and I did 3 times API and got the same results for them. as you mentioned before I think Staphylococcus can be adapted to these food sources and gives a similar profile as lactobacillus specially in the marine samples. (which is not easy to isolate "typical" LAB from them and in some studies they classified "atypical" lactobacillus for these samples). if I do again sequencing I think will get same results as before because no one in lab working with Staphylococcus and also my isolates were fresh. ( the BLAST results for Seq of Staphylococcus was 98%) do you have any idea to inhibit growth of Staphylococcus on MRS media? MRS should be selective for lactobacillus, pediococci, leuconostoc so how can increase selectivity for example only for lactobacillus and inhibit undesired organisms like Staphylococcus and sometimes Streptococci even if they are not from cross-contamination? I did some modification on MRS agar and incubated the plates under anaerobic condition but I think is not sufficient for this case...!?
Leo
Leo
Maybe there are some bacterial-taxonomists here who can help me out, but as far as I remember 98% similariy of the 16S is not very high! I think to remember that 98/97% is even discriminating between two species (but I`m no expert on bacterial taxonomy...)
The second problem is: API is usually tested and evaluated for clinical or some food applications. With "strange" bacteria form complex environments you have a high probability to get wrong results...look for the Yersina example above!
have you done a good old gram-staining? Maybe this helps you out!
As I understand your post, you used MRS medium all the time? Try to cultivate your bacteria on non-selectiv media (TSA or similar) to get pure cultures and to avoid "repressed" growth of Staphylococci etc...
look at the microscope again, check for endospores, size, motility....
If the proper ID of this isolate is very important for your work, I am afraid the only thing you can do is to try to find out in an old fashioned way: just taking Bergey's Manual and look into which taxonomic unit your strain can be sorted....
g
Hey there,
May I point you to this:
http://jcm.asm.org/cgi/content/abstract/44/4/1359
Although its for non-fermenting bacteria, the criteria used for 16s rDNA identification (under the methods) is stated there. If its >99% then the unknown is assigned to that reference species. If its between 99% and 95% as in your case, then it is assigned to the genus of the reference bacteria.
I'm completing my dissertation on isolation and identification of bacteria and bumped into some discrepancy between the 16s rDNA and the biochemical. Well, I didn't use the API kit but did it manually. If you can get your hands on a plate or two of mannitol salt agar then you could see if its Staph or not. Could be prepared if you have the resource and time
http://www.bd.com/ds/technicalCenter/inser...l_Salt_Agar.pdf
Neway the Gram stain should discriminate on the basis of shape. Use a fresh culture ok?
Most of the time I used MRS medium to cultivate this bacteria but I also used Nutrient broth during overnight incubation and observed some haven't growth and some just got a small growth and then I kept it for longer incubation period but still same and/or not much growth. (is it because of slow metabolism of LAB compare to other bacteria!?) at the same time and same condition the culture on the MRS broth grown well even after 24 hours 37 C. on TSA broth I have tried once and it grown but haven't checked for microscope, endospores, size, motility ... as you said. (I will try it again,Thank you)
There is one thing regarding to forward and reverse primers that I used. I don't understand what does it means in the results that received mentioned "Early Single Loss" !? I got 2 chromatogram and seems they are good (not noisy) but for one of them "effective length of query" is 363 and for an other is 559 !?
Actually it was my first time that I did 16S and I'm not expert in this area. I just borrow the primers from other lab and I don't know whether I used suitable primers or not? suppose if next time I want to do again 16S, as a unknown bacteria should I used the Universal Primers? is it need to know the sequence of this primer before I order it? how can get the DNA sequence of it? do you have any idea about it?
Leo
See the articles linked here. The Lane91 primers listed there are the standard bacterial universal primers.
http://openwetware.org/wiki/Bacterial_species_identification
i did screening on my bacteria isolated from different host... using both biochemistry and 16S.
I also did conventional and API for biochemical identification...
what i can say is: DNA identification (using 16S or other conserved gene) is more accurate then biochemical test. Biochemical test only served as a support to what you have for your DNA test.
different strains might have some differences in using the chemical/sugar provided in biochemical test. sometimes also depends on how much culture you put in... if you put less, you might not see the difference; sometimes, the changes is not obvious and you might need soemone who is experienced to interprate the result.
for me, it is not surprising if you get difference in biochemical test compare to DNA identification. Even though sequencing 16S or other conserved genes will still give you error... the result still more reliable compare to only biochemical test.