MLST is used for tracking MRSA, E.COli strains etc, for which there is lots of isolate data, genomic data and clinical data.

If you are working on exotic novel strains it would not help.

Reliability is more of the question and context, 16 works fine for some bacteria but can fail miserably for Bacillus for eg.

So if you know what you are looking for, you can use markers that better help delineate them to species/strain level which might not necessarily be 16S or MLST.

Thanks Jay, but my concern is that the housekeeping genes (MLSA) have many advantageous over 16S like they are conserved and have protein products, so more reliable than 16S. 

For Pseudomonas novel strain prediction if MLSA helpful? to resolve it up to the level of species because 16S is good but up to the genus level. 

Among Pseudomonas strains yes. MLST is helpful, again depending what you are looking for, context of the question.

http://pubmlst.org/paeruginosa/

No because there are a lot of ST that are not already described so in those cases it would be impossible the identification

I do think that the MLST analysis is helpful for identification of strains up to species level.

I have been working with MLST sequences for Pseudomonas this passed summer. Although my P. fluorescent strains have identical 16s sequences (amplified by 27F and 1492R), but they appear to have different phenotypic characteristics, And MLST analysis does show that they have difference in sequences. Some of strains are so closed related that they have identical sequences when I align 4 different MLST sequences + 16s rRNA sequence but they showed difference when I put other 3 MLST sequences to compare. 

And in general, for routine laboratory techniques, sequencing 16s would be quicker and more useful and sometimes you don't need to sequence the whole 27F & 1492R sequences.

i agreed Nguyen and have experienced same as you said that is the reason i am focusing on MLSA for Pseudomonas identification 

Dear Maria, 

Agreed all ST not describe so for but if do with known about 7 to 12 MLSA genes then i think it helpful for species discrimination and identification up to level of species as compared to 16S rRNA.

This marker gene (16S rRNA) is highly conserved and universally present in bacterial strains. The lack of variability of the 16S rRNA gene sequence did not allow identification of closely related species or subspecies.(Hung et al., 2005).Most bacterial genomes contain multiple copies of the 16S rRNA gene, and copy number varies per species, This is further complicated by the fact that sequence variation between the different 16S rRNA gene copies present exists in some genomes 

In contrast to DNA–DNA hybridization and 16S rRNA gene sequence analysis, multilocus sequence analysis (MLSA) is capable of yielding sequence clusters at a wide range of taxonomic levels, from intraspecific through the species level to clusters at higher levels (Gevers et al., 2005).Housekeeping genes typically occurs in a single copy. The high level of conservation across 16S rRNA genes can obscure most intraspecific, and sometimes interspecific variation. In contrast, the higher resolution housekeeping genes marker is capable of revealing molecular variation down to the population level.

So in line of these findings, we may used MLSA in place of 16S for Pseudomonas identification up to the species level.???

I never think about it but when I try to BLAST the MLST sequence of one of my isolates, it does give the information up to species. So by all means, MLST can be used for Pseudomonas identification up to species level.

But one question is what makes you think of replacing 16s by MLST in your research?

Dear Nguyen,

Why i am in favor of MLST in place 16S, the answer i already coated in my above comment where clear show the impact / importance of MLST over 16S.

But if you feel this is not true then please elaborate your point of view in reply to my above comment which may be helpful for me. Thanks

I'm sorry, it was my bad for not reading your points carefully before asking questions. I can see the advantage of having the comparison among closely related species with MLST as well as using MLST sequence for identification. A lot of time the 16s rRNA sequences don't give you the information down to species level.

It is all right and yes MLST seem better than 16S according to my experience and will be used for identification up to the species level but need point of view of more and more peoples in this regards. 

Although this topic has been thoroughly discussed I wanted to add my 2 cents.

Your initial question Muhammad is a little misleading since you ask it generally but later restrict it to a single genus.

Within a genus you might use a MLST/housekeeping but as Jay already pointed out this does not allow analysis of unknown samples and even less the identification of novel species outside your specific group of interest. The identification of novel bacterial species is quite tricky anyways. This is why microbiologists came up with the OTU, witch is actually based on 16S.

Given the specific case a MLST might be better suited but including 16S as additional locus would provide additional confirmation with comparably low effort.

Cheers,

Manuel

Dear Manuel,

Thanks for your valuable comment but please tell me what is the best and cheapest molecular method to identify the new strain case else than 16S, MLST and DDH?.

Given your specific case, working in Pseudomonas, MLST would definitely be the more appropriate approach and my first choice!

Yes to extent it is good for strain identification in a very few organisms. Ultimately alignment of 16rRNA sequence will be more a positive answer.

Dear Jai,

How 16S is better in line of these findings?

This marker gene (16S rRNA) is highly conserved and universally present in bacterial strains. The lack of variability of the 16S rRNA gene sequence did not allow identification of closely related species or subspecies.(Hung et al., 2005).Most bacterial genomes contain multiple copies of the 16S rRNA gene, and copy number varies per species, This is further complicated by the fact that sequence variation between the different 16S rRNA gene copies present exists in some genomes

In contrast to DNA–DNA hybridization and 16S rRNA gene sequence analysis, multilocus sequence analysis (MLSA) is capable of yielding sequence clusters at a wide range of taxonomic levels, from intraspecific through the species level to clusters at higher levels (Gevers et al., 2005).Housekeeping genes typically occurs in a single copy. The high level of conservation across 16S rRNA genes can obscure most intraspecific, and sometimes interspecific variation. In contrast, the higher resolution housekeeping genes marker is capable of revealing molecular variation down to the population level.

So in line of these findings, MLSA seems to be a better choice than 16S.

1) MLST works well for things you know and have been well defined like microbes cultured and characterised in labs.

2) you can have sets of universal primers that can amplify a large percentage of microbes its not same for MLST.

3) Housekeeping genes also get duplicated and have variants depending on the evolutionary pressure eg: gyb rpob etc.

4)There is an extensive database and information for 16S but not so for MLST, which is restricted to strains of clinical or industrial relevance.

Dear Jay,

Thanks for sharing good points regarding query but  I read few recent papers on MLST, where they clearly showed dominance over 16S and please elaborate your point number 3 or refereed me some literature regarding the duplication and variant of gyrB and rpoB.

here is the search I used

"gyrase b variants in clinical isolate"

and typical result is

http://aac.asm.org/content/37/4/696.full.pdf

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0056477

I am sure you can search for similar articles and find answers relevant to your question.

As many others have pointed out in the course of the discussion, MLST does have its advantages in the clinical environment but for poorly described species its not a good idea to deploy.

Thanks Jay

16S sequencing will speciate. MLST or MLEE does not differentiate species, rather they differentiate within a species. Beware, MLST only analyzes for neutral housekeeping genes not under diversifying selective pressures, thus misses variability typical of the remaining 99% of the genome, thus not possible with MLST to determine whether two strains are identical. Both 16 gene analysis and MLST are reliable, each based on DNA sequencing. More relevant questions is, what is the question that you are addressing?

Dear Goldstein, agreed with your comment, my question now is that is the MLSA better/more reliable than 16S for identification/differentiate between the species of Pseudomonas genus?