We cultivated unknown bacterial species from a clinical specimen. Result of the 16S sequencing were two species of Asaia - A. siamensis and A. bogorensis. Who can give me some tips how to differentiate between this two closely related species?
A quick pubmed search teaches that currently the only distinction is the ability to produce acid from dulcitol that is only found in A. bogorensis http://ijs.sgmjournals.org/content/51/2/559.abstract?ijkey=037b8a32fecba19653caf89e319e8128b5b19a3a&keytype2=tf_ipsecsha.
I'm not exactly sure if I'm understanding correctly, as it seems that you have already distinguished between the two isolates using 16S sequencing. Using a genomics based approach, you could try performing SNP analysis if you can sequence the whole genome, or perhaps use T-RFLP or an RFLP based approach.
You can distinguish based on rpoB gene(~730bp) [or groEL gene (~945bp), dnaK gene (~788bp)] sequencing. {JOURNAL: Int. J. Syst. Evol. Microbiol. 60 (PT 10), 2277-2283 (2010) Title: Phylogeny and differentiation of species of the genus Gluconacetobacter and related taxa based on multilocus sequence analyses of housekeeping genes and reclassification of Acetobacter xylinus subsp. sucrofermentans as Gluconacetobacter sucrofermentans (Toyosaki et al. 1996) sp. nov., comb. nov. http://ijs.sgmjournals.org/content/60/10/2277.full?sid=595c9897-0b9e-48cf-80ef-bfc5645afbb4 }; I wish you success.
Thanks to all of you! It helps me a lot.
Dear Brian we have one clinical isolate identified according 16S sequencing as A. siamensis or A. bogorensis. I just want to know which one from this two options is right . Mainly because this bacteria are rare in human clinical samples.
I see. We have a similar issue with 2 species we work with. They are indistinguishable by 16S (>99% homology). I am not familiar with your organisms in particular, but I would agree with Fwu-Ling Lee above. We also look for unique genes to distinguish the 2 species. Perhaps developing a PCR-based assay targeting those unique genes Lee identified above will solve your problem.
More concrete answer: IF the rpoB gene sequencing results showed that your isolate approximately had > 98.2% sequence similarity to the type strains of Asaia siamensis, and < 89.5% sequence similarity to the type strains of Asaia bogorensis, your isolate should be Asaia siamensis. The pimers for rpoB, dnaK gene were listed in this literature [http://ijs.sgmjournals.org/content/60/10/2277.full?sid=595c9897-0b9e-48cf-80ef-bfc5645afbb4]. Sequencing methods are also described in the literature. IF the dnaK gene sequencing results showed that your isolate approximately had > 98.2% sequence similarity to the type strains of Asaia siamensis, and < 92.7% sequence similarity to the type strains of Asaia bogorensis, your isolate should be Asaia siamensis.
Interpretation and vice versa.
The "multilocus sequence analysis" (MLSA) method is often used by bacterial taxonomists.