Have you tried the Gas chromatography method, it detects cell wall fatty acids that are genetically concerved, it is not a DNA based method. The system is the Sherlock system by MIDI. http://www.midi-inc.com/

16S rRNA gene sequencing.

I think so, 16S rRNA gene sequencing.

The most specific is rRNA sequencing.

I suggest the following methoids: rpoB Gene Sequence-Based Identification of Aerobic Gram-Positive Cocci of the Genera Streptococcus (Drancourt et al. 2004, Journal of Clinical Microbiology vol. 42 no. 2 497-504) or Amplified Ribosomal DNA Restriction Analysis (ARDRA) followed by 16S rRNA gene sequencing on distinct ARDRA groups (Outtara et al. 2011 Food Microbiology Volume 28, Issue 1, February 2011, Pages 1–8).

My colleagues at the CDC Streptococcus Reference laboratory use both 16S rRNA and rpoB gene sequencing and feel very confident about the ID of Streptocccus species.

I understand what you mean, but I am not aware of another good gene, besides what you have listed and perhaps rpoB, to target. It is becoming difficult to get most microbes down to the species level - especially Bacillus spp.. Even full 16S is no certainty when identifying with this gene only. I am not sure on fatty acid profiling - not something Ihave seen much of (is this just variation on lipid analysis)?

16S rRNA ribotyping

I think 16S rRNA and rpoB are the best.

With regards bacillus; PCR for the genomic target and the two plasmids (cap and lef)

real time PCR

16S rRNA gene sequencing

bacillus on blood agar hemolysis beta colonies large gray and strep diagnosis by strip stre API AND colonies small catalase -

For this type of bacteria, I strongly suggest 16S rRNA gene sequencing. I discourage proteomic tools as MALDI.

Sequencing of gene "tuf"is also a good approach for Streptococcus identification.

pulsed field gel electrophoresis (PFGE), Multiple Loci VNTR Analysis (MLVA), Multilocus sequence typing (MLST) and (multiplex-) PCR-based methods.

Further to J Viponds reply: see La Duc et al., 2004 J. Micro. Methods 56(3) ,"gyrB as a phylogenetic discriminator for members of the Bacillus anthracis–cereus–thuringiensis group", and for interest Qi et al., 2001 AEM 67(8) 3720, "Utilisation of rpoB gene......".

It is great to read such a precise requirement. I am not sure of the detail you want to achieve. For the documentation of a novel species refer to the International Journal of Systematic and International Microbiology. 16S RNA sequencing will group the bacteria, thereafter the sequencing of selected conserved genes i.e. ddl, rhoB and sodA genes in the case of the streptococci. If these are not satisfactory - you will have to undertake further investigation.

If you know what you are dealing with, the detection of specific genes by QPCR is quicker than sequencing. Microarray technology albeit expensive may assist you in multiple gene detection.

go for biochemical tests as IMViC,catalase activity and further gene sequencing

Thanks to everyone for their replies, its great to see some many responses, which i'm sure is going to keep me busy!

In all honesty I agree with the comment by Patrick Druggan, my request is somewhat double edged - meeting the actual needs of the clinician while 'developing' faster, better (higher res) methods to 'better?' meet their needs. 16s works well and is arguably the gold standard by which new rapid id methods are measured, however it does have drawbacks in its ability to resolve certain groups for example: E. coli/Shigella, the B. subtilis group, B. cereus group and the Streps. It with MALDI are being used for unknowns and for generating presumptive ids where the exact species isn't known, but we do get requests from clinicians for a more accurate identification, for example is it B. cereus (more likely) or B. thuringiensis (less likley - insect pathogen) or with E. coli vs Shigella spp. etc., but we do get the requests. Hence my question, but I accept the point is the greater resolution actually needed?!?!

I guess by asking what the m/o is, you assume the rest, virulence factors - toxin production, spore former, transmission and antimicrobial resistance (although all these factors can be tested for) - its true its a bigger question than i first thought!

I am curious about the fatty acid GC method i've not come across the commercialised ID system, i've been looking for recent publications which have targeted the Bacillis or Streps but haven't had much luck, if there any recent publications could someone list some here, that would be very much appreciated!

Thanks again to everyone!

Bacillus species used for microarray analysis in this study were presumptively identified by subculturing 1 μl of each broth (used to produce DNA for PCR analysis) overnight at 37°C on Columbia blood agar (Oxoid). A further 1 μl of each culture was diluted in 10 μl of water and each sample was spotted onto a microscope slide. Slides were fixed in 100% ethanol for 20 s and gram stained (Sigma Gram Stain). Bacilli were viewed under the microscope and the size and morphology of each isolate were recorded; strains were subsequently grown in nutrient broth containing 0·001% (w/v) lysozyme (Sigma Chemical Co.) in 0·1 mol l−1 HCl diluted 1/100 in nutrient broth (Oxoid) at 37°C for 14 days. Isolates were also plated onto prereduced Columbia blood agar plates and incubated under anaerobic conditions for 5 days at 37°C (Parry et al. 1983).

Thanks Raju

The resolution required is dependent on the aims and objectives of your laboratory and how critical the identification is. For instance you may find that rapid detection of B. cereus enterotoxin is all that is required. That the strepAPI or Biolog system is good enough for the streps etc.

I think that the approach of Maiden group based on variation of the 53 genes encoding the bacterial ribosome protein subunits could be usefull:

http://www.ncbi.nlm.nih.gov/pubmed/22282518

I believe also that the 16S r RNA Ribotyping is one of the best approach for rapid diagnosis

If one has clinical and microscopic acumen the Gram's stain is the most simple, accurate and quickest method. For example if one is looking for sputum specimen then likely pathogens morphology is known such as H influenzae, Str peumoniae, K. pneumoniae, and so on. Expensive and time consuming Molecular methods are unnecessary for routine work. However on could use when it is required.