First, there are no clear (official) rules on species/subspecies determination. So you'll have to go along with the current determination of the species/subspecies in the genus you're interested in.

Second, whether your isolate belongs to a certain species or subspecies depends on the results of different tests and depends on the genus. In Deinococcus, 16S rRNA gene sequencing will satisfy to characterize the isolate at species level. For others you could use MLSA, MLVA or even ANI analysis. These will resolve the isolates of the genus to a certain level, at which point you might have to consider to change the current opinion on species/subspecies classification in that genus.

From my experience, I believe that MLSA or MLVA can do the trick, but again, that depends strongly on the genus of interest. If you have plenty of resources you could do whole genome sequencing and run an ANI analysis. Other tests like PFGE, RAPD, AFLP, RFLP or REP-PCR can also help you but their resolution is generally lower than MLSA or MLVA. MLSA is easier to design but slightly more expensive than MLVA.

If possible, you may try multi-gene analysis. This will give you a valid discrimination among the subspecies.

Guido Cordoni has you on the right track. However, if the genus or specie are unknown at this points, you need to start with a 16S to get you into the ballpark. I had the same issue with marine Vibrio sp. Sometimes it takes multiple genes. Good luck.

Several molecular strategies can be conducted to discriminate bacterial, fungal or protozoa phenotypes. The sequencing of the 16s SRNA coding fragment can be of great help. Moreover, the High resolution melting after real time PCR is a useful tool to draft species specific profiles. If these strategies do not allow to specify species or intraspecies profiles, the analysis of mitochondrion sequences can add other elements that will help to build profiles and draft phylogenetic trees, that interesting, are not always equivalent or overlap those obtained with the analysis of the 16 s SRA sequences.

It will depend on the genera/specie. But, you should try 16S rDNA, 23S rDNA and girB. These multi-gene evaluation usually give you a good discrimination.

First, there are no clear (official) rules on species/subspecies determination. So you'll have to go along with the current determination of the species/subspecies in the genus you're interested in.

Second, whether your isolate belongs to a certain species or subspecies depends on the results of different tests and depends on the genus. In Deinococcus, 16S rRNA gene sequencing will satisfy to characterize the isolate at species level. For others you could use MLSA, MLVA or even ANI analysis. These will resolve the isolates of the genus to a certain level, at which point you might have to consider to change the current opinion on species/subspecies classification in that genus.

From my experience, I believe that MLSA or MLVA can do the trick, but again, that depends strongly on the genus of interest. If you have plenty of resources you could do whole genome sequencing and run an ANI analysis. Other tests like PFGE, RAPD, AFLP, RFLP or REP-PCR can also help you but their resolution is generally lower than MLSA or MLVA. MLSA is easier to design but slightly more expensive than MLVA.

16S rDNA will provide you, as Mike mentioned, a ball park; probably down to the genus level. Once you have acquired a genus-level ID, 16S rDNA compatibility can be combined with DNA-DNA hybridization of species related within that genus. The two approaches combined provide a greater taxonomic resolution beyond either one individually (Rossello-Mora & Amann, 2001). However, if you want to identify beyond the species level, down to the strain, other molecular techniques and biochemical testing will be necessary. Serological testing and sequential biochemical dichotomies will be very useful.

First I would go for 16S rRNA sequencing it can give you an option for the following step like RFLP, PFGE, RAPD, AFLP.

You can also subculture the bacteria for pure culture and try some biochemical tests then use the standard scheme for bacteria identification.

Good luck

As everyone else have pointed out, you shouldn't rule out sequencing the 16s rDNA gene since it will help you to locate your bacteria in the genus they belong. However to achieve identification up to species or subspecies levels you should sequence at least four more genes to have a certain degree of certainty. Generally, rpoD, rpoB and gyrB have been reported to discriminate up to species and even subsp level, but it depends on the bacterial genus you are dealing with.

On the other hand, you can complement the molecular approach with some basic biochemical and microbiological tests (oxidase, Gram stain, carbon source assimilation, etc.) contained in some kits as the BIOLOG or the API, since physiological traits are also important when identifying a bacterial strain.

Best of luck!

16S rDNA sequence analyses and DGGE fingerprinting are appropriate methods for the detection and identification of monomicrobial as well as polymicrobial infections of bacteria that might not be detected by conventional cultivation. Fragments of 200 bp, spanning the V3 region of the eubacterial 16S rDNA, should be amplifie by poly- merase chain reaction (PCR) and separat by denaturing gradient gel electrophoresis (DGGE). For phylogenetic identification, DGGE bands should be excise and directly sequenced, or 16S rDNA clone libraries should be construct and clones should be screene by DGGE. Sequences should be compare with sequences of known bacteria listed in the EMBL database.

In addition to 16S, you could tie in data from conventional biochemical testing panels, for example API test strips. Neither is going to be 100% definitive, however together they may give greater confidence of identification:

http://www.biomerieux-diagnostics.com/servlet/srt/bio/clinical-diagnostics/dynPage?doc=CNL_PRD_CPL_G_PRD_CLN_11

That study is a bit old nowadays. I recommend you to read about the different regions in the 16s gene and about pyrosequencing (454 sequencing). Still I agree that some genera can be more difficult than others. Where are you trying to identify communities from? environmental samples? which kind? We may need more information to better cover your question. Cheers, JPM.

Have your isolate's 16S rDNA gene sequenced. BLAST the sequences to determine to which genus it clearly belongs. Now do phylogenetic studies by aligning your sequence to all the Type strains of the species in the genus. Your bootstrap values should tell you to which your isolate is closest to. However, if you have a percent similarity less that 95% then this is a candidate for a novel species. DNA:DNA Hybridization should be employed in this case.

Physiological, Biochemical and Chemotaxonomic Tests should also be done for validation of your genetic work. :)

As many pointed out above, do the 16S rRNA (Bacteria/Archaea (18S for Eukaryotic species) it is the start, then do as Tabao lined out. But importantly, go to IJSEM (former IJSB) and look for the minimal description of the genus / group for possible discriminating assays/tests, get the original descriptions of the 5 or so closest species (i.e., do a thorough literature study (including but not restricted to the description in Bergey's Manual) then do the required / suggested biochemical physiological test.

The concept of subspecies is a problematic one and only used in some groups in others it is more regarded as a chemovar etc. ~var. Some species have a relative wide variation in their described properties (see first descriptions of taxa where many strains were isolated). Keep in mind there is no general or official rule what is a subspecies and what is an unusual strain of a species. Many times it depends on the tradition how scientist handled the specific microbial group.

Thank you all for your valuable suggestions. We have done 16S rDNA sequencing and will take into account the other techniques that can be used to resolve the identity of the bacteria we have isolated.

An additional technique not mentioned yet would be mass-spectrometry based analysis like MALDITOF.

One of the best things that you could do is reading the article:

Notes on the characterization of prokaryote strains for taxonomic purposes.

Tindall BJ, Rosselló-Móra R, Busse HJ, Ludwig W, Kämpfer P in Int J Syst Evol Microbiol. 2010

They are well know taxonomist and give a clear reference in these article of all the things you need for describe a novel species.

In general, 16S rRNA is important, but there are a lot of other things that you have to check (DNA-DNA hybridization, chemotaxonomy, phenotipic differences...)

16S RNA gene amplification works quite well for this case, it's what we do all the time.

As everyone else have pointed out, you should use sequencing the 16s rDNA gene in order to locate your bacteria in the genus they belong. then to achieve identification up to species or subspecies levels you should either do PCR specific for each spcies under this genus or sequence at least four more genes to have a certain degree of certainty. Generally, it depends on the bacterial genus you are dealing with.

On the other hand, you can complement the molecular approach with some basic biochemical and microbiological tests (oxidase, Gram stain, carbon source assimilation, etc.) contained in some kits as the BIOLOG or the API, since physiological traits are also important when identifying a bacterial strain.

An additional technique would be mass-spectrometry based analysis like MALDITOF. if your organism present in the database.

you can also use th molecular tools for genetic differentiation under on specis level o what is calld genotyping methods; PFGE, MLST, Microarray, AFLP, ......

Best of luck!

I would use not only 16S but also 23S and the inter-space region , a complete sequence can then be blasted and give you a better idea. If that does not resolve completely, I would use an additional gene marker such as rpoC1. If you have the bacterium isolated in cultures then no need for DGGE.

To be able to identify novel bacterial species and to have the novel species published, I would suggest to go to the IJSEM to find out the standards to publish a new species. The most important is to determine the bacterial genus and to compare the new strain with the type strains of the other species from the same genus. I would first test phylogeny of MLSA data, or, if the genome sequence is availabe there is also an in situ hybridization which you can find online, if now genomic data avialable try first Biolog test.

If available for your genus of interest, do MLSA. As an example, for vibrios, we usually do 16S to get an idea, many times is all we need for certain clades, but for other clades, we sequence one or two housekeeping genes like pyrH or topA and that gives us a good identification. In my experience, phenotypic test are now a waste of time for identification most of the time.

Hi everyone. Please can you suggest any basic introductory book for a beginner on molecular methods of bacterial identification? Where I am from, the traditional methods are still used but i want to learn the modern methods. Thanks.

16S rDNA sequence analysis after that perform DNA-DNA hybridization with most closely related species (type strains)

IMHO, the method of choice is MALDI/TOF mass spectrometry...

With MALDI/TOF-MS you are lucky if you can discriminate up to the genus level. In a few cases, species identification is possible, but don't expect to differentiate subspecies.

Like Rehab El-Shehawy suggested above, you get a better idea by adding more sequence data, such as 23s, but I also suggest that you add your sequences to an alignment of the same regions in known bacteria and that you then use this matrix to run some phylogenectic analyses.

In a lot of analysis, scientists consider a percentage of similitud, for example De Clerck (2004) with sequence similarity of

The best is to do polyphasic taxonomy. I mean a combination of 16S, Biochemical method and Fatty acid profiling (FAME) confirms you to speces level.

The 16S rRNA sequencing is only used for the identification of different bacterial strains ,i think MALDI/TOF-MS is the better option for identification in species or subspecies level.

16s RNA is a phylogenic gene so that you can use this tract for identifying lots of microorganisms in the level of species if you use sequencing method, otherwise there is no specific primer for the identification but some few species. My advice is use of a single or more restriction enzyme for cutting your amplified DNAs in rRNA gene to do discrimination of some more species, if you try to find the best enzyme/s which be able to make an appropriate differential pattern.

There are lots of techniques with more or less phylogenetic resolution. Attached paper has a nice figure to help to figure it out.

¡Saludos!

you could try ribotyping http://foodscience.cornell.edu/cals/foodsci/research/labs/wiedmann/lmt/about-ribotyping.cfm

A multilocus sequence analysis approach with several housekeeping genes should be the better way. It is highly discriminative for difficult genus such as Vibrio, Aeromonas or Pseudomonas. There is plenty literature about it.

Species identification is possible by the use of universal primers together with high-resolution melting analysis. This is explained in the links below :

http://jcm.asm.org/content/47/7/2252.full

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2562909/

MLST is a very good option, I wouldn't reccomend MALDI-MS as above as there are significant problems of intra-species i.d., in particular with enterics.

there are several ways.

first you do primary tests like- Grams staining, motility test, capsule staining etc

then refer Bergey's manual and carry out biochemical tests. or go for 16s rDNA

Thanks a lot, dear Sebastian F Alburquenque, for the article.

Routinely, biochemical tests are the first and simple methods; however, fatty acids profiling or molecular based methods such as ribotyping, 16S r RNA and MLST analysis have high discriminative power. Although, some prefer using several of these techniques together.

Hi,

I think for IDing to the subspecies is the same as saying strain typing. Example, is your E. coli DH10B or DH5Alpha. Strain typing may be done using Bergies manual or RFLP is used.

In molecular biology, restriction fragment length polymorphism, or RFLP (commonly pronounced “rif-lip”), is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites, and to a related laboratory technique by which these segments can be illustrated. In RFLP analysis, the DNA sample is broken into pieces (digested) by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis. Although now largely obsolete due to the rise of inexpensive DNA sequencing technologies, RFLP analysis was the first DNA profiling technique inexpensive enough to see widespread application. In addition to genetic fingerprinting, RFLP was an important tool in genome mapping, localization of genes for genetic disorders, determination of risk for disease, and paternity testing.

I agree with M. Moreno but not with RFLP, rep-PCR is a cheaper, faster and highly discriminative way to fingerprint strains of bacteria (maybe not to identify above species level) but certainly below species. There are several primer sets that work with different bacterial groups (REP, ERIC, BOX, GTG5). This is a fast approach that can be latter complemented with MLSA, as Jesus Romalde said.

Microbial Identification System is ideal for the issue

Your best bet is to use an identification protocol that best suits your situation

1. What bacterial species does you laboratory or project have to identify - medical pathogens, specific families etc i.e. You wish to identify shedders of Salmonella - to do this you will have to identify potential salmonellae to species, serotype, genotype and phagetype level.

2. What bacteria is a basic identification sufficient i.e. a research project is only interested in pseudomonads, but the selective media used will allow bacteria that are not pseudomonads to grow. Therefore all you will need is a simple test i.e. Gram's stain and oxidase test or glucose fermentation test to exclude them.

3. What skills and instrumentation are available? phenotypic as opposed to genotypic identification

4. Funds available

Stating that: Gram's stain, oxidase. catalase, motility and sugar fermentation tests should always be your base-line tests as they help in assigning bacteria to groups and avoid some mistaken identifications when using for sophisticated tests.

For manual identification of medical related bacteria the best reference books are

Cowan and steel: ID bacteria

Koneman: Diagnostic microbiology

Bailey and scott: Diagnostic microbiology

Bergey's manual of identification of bacteria may be useful for identification of bacteria to species level

Do 16s rRNA gene sequencing and their phylogenetic analysis.

1. Use (almost) complete sequences

2. Check the quality of new and reference sequences: ambiguities, primary and secondary structure consensus violations, overlay of potential cistron heterogeneities (direct PCR fragment sequencing)

3. Remember that the sequence databases are full of incorrectly labelled and poor quality sequences. There is NO justification for using a sequence of poor quality/dubious origin simply because it is in the database. When characterizing new taxa, a taxonomist should use the best quality data available, including resequencing if appropriate.

The use of expert-maintained seed alignments comprising only high quality sequence data is highly recommended, e.g. ARB (www.arb-home.de), RDP (http://rdp.cme.msu.edu/), SILVA (www.arb-silva. de) and LTP (www.arb-silva.de/projects/livingtree/). The European rRNA database at the Department of Plant Systems Biology, University of Gent, is no longer updated (as of February 2007) but a link is provided to the SILVA website. A limitation is that seed alignments may not be universally compatible with some of the programs used by authors of articles in the IJSEM

4. Pairwise nucleotide sequence similarity values should be calculated with caution. The following should be considered:

(i) the way in which the similarity was calculated should be given in detail. The following programs are recommended for similarity calculations: ARB, PHYDIT and jPHYDIT. Programs such as CLUSTAL or PHYLIP give the dissimilarity values. EzTaxon (www.eztaxon. org) provides a web-based tool.

(ii) pairwise similarity values obtained from local alignment programs, such as BLAST and FASTA, should not be used. These programs are primarily useful for database searches.

(iii) corrected evolutionary distance (e.g. Jukes and Cantor model) should not be used for pairwise similarity calculations.

(iv) full-length sequences should always be used.

5. 16S rRNA gene sequences alone do not describe a species, but may provide the first indication that a novel species has been isolated (less than 97% gene sequence similarity). Where 16S rRNA gene sequence similarity values are more than 97% (over full pairwise comparisons), other methods such as DNA–DNA hybridization or

analysis of gene sequences with a greater resolution must be used. These methods must also be correlated with the characterization based on phenotypic tests. While the resolving power of the 16S rRNA gene with respect to the delineation of

novel species has been extensively debated, less attention has been paid to other taxonomic ranks, such as the genus

6. At values above ~95% 16S rRNA gene sequence similarity (over full pairwise comparisons), taxa should be tested by other methods to establish whether separate genera are present

6. DNA–DNA hybridization (DDH) is to be performed in cases where the new taxon contains more than a single strain, in order to show that all members of the taxon have a high degree of hybridization among each other. DDH is necessary when strains share more than 97% 16S rRNA gene sequence similarity.

7. cultural, morphological and phenotic characteristics of type strains are also taken consideration to assign a sp or sub sp.

Identification of an isolate is a dynamic process, so there is very difficult to establish unique method for isolation of all taxa. Also, I have to emphasis that identification rules are varied during the time. So, analysis of 16SrRNA gene sequences only is a preliminary test to show the rout of finding the roadmap of identification. Then, you have to study the relative chapters in Bergey’s Manual of Systematic Bacteriology. For example, analysis of 23 SrRNA gene sequences, chemotxonomy (sugar and amino acid of cell wall, phospholipid of cytoplasmic membrane and ….) are differentiative for one taxon and are not for others. Finally, you can consult with a taxonomist of relative taxon or read relative article in International Journal of Systematic Microbiology.

First confirm your strain using whether new or not using 16 rDNA sequencing. If

If you need rapid answers there is a rapid, sensitive and specific test able to inform on: Bacteria, and if yes how many(CFU/ml). In addition, this test confirms simultaneously signals for: Gram positive Cocci, and if yes, how many (CFU/ml); Staphylococci, if yes how many,Streptococci; Pseudomonaceae; Propionibacteria; Corynebacteria sp.; Enterobacteria; Acinetobacter sp; Haemophilus sp and Stenotrophomonaceae.

Several species may produce slime or other extracellular sticky matrixes. Therefore, ultrasound treatment should be used to release and suspend germs that bind to the support before DNA extraction. The ultrasound conditions (intensity and time) should not alter the viability of Bacteria. Signals for viable and no- viable Bacteria are obtained in less than 2 hours.

Hereafter some references for the procedures:

Goldschmidt P, Balloy T, Degorge S, Benallaoua D, Batellier L, Koskas F, Borsali E, Chaumeil C. New ultra rapid test for detection of bacteria. Pathol Biol (Paris). 2009 Nov 24

Goldschmidt Pablo, Sandrine Degorge, Djida Benallaoua, Elena Basli, Laurence Batellier, Sandrine Boutboul, Cécile Allouch, Vincent Borderie, Laurent Laroche, and Christine Chaumeil. New test for the diagnosis of bacterial endophthalmitis.Br J Ophthalmol 2009;93:1089-1095

Goldschmidt Pablo, Claudia Costa Ferreira, Sandrine Degorge, Djida Benallaoua, Sandrine Boutboul, Laurent Laroche, Laurence Batellier, and Christine Chaumeil. Rapid detection and quantification of Propionibacteriaceae. Brit J Ophthalmol. Vol: 93,2: 2009: 258-63

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1st of all u need to do 16s rRNA gene sequencing for phylogenetic analysis, basing on that u can confer whether ur bacterial strain belong to which genera and then for confirmation as having novelty u can do group specific biochemical, physiological, enzymatic characterization, G+C content anlysis, total cellular fatty acid profiling, respiratory quinones profiling, DNA-DNA hybridization then only u can say ur strains possesses some novelty...

Species ID can be done both by conventional methods and by other molecular methods. I depends on whether we can afford to do

Sometimes biochemical identification is enough.

Hi dear,

16S rRNA analysis not enough to identified bacterium at species level, in this concern you may go for housekeeping gene( rpoB, rpoD,recA) identification which help out to identified the species

The use of the rpoB gene offers various potential advantages over standard 16S rRNA gene-based approaches. First, since most bacterial genomes contain multiple copies of the 16S rRNA gene, and copy number varies per species, rpoB typically occurs in a single copy

Second, the high level of conservation across 16S rRNA genes can obscure most intraspecific, and sometimes interspecific variation. In contrast, the higher resolution rpoB marker is capableof revealing molecular variation down to the population level

Third, genetic divergence of rpoB correlates better with overall genomic divergence and provides better bootstrap support for phylogenetic reconstruction.

Fourth, given the fact that rpoB is a protein-encoding gene.

For detail refer to Vos et al., (2012).

RE M. Naveed's comment:

Just to avoid misunderstandings: You need to do housekeeping genes sequence analysis in connection with 16SrRNA gene analysis. You need to do more than just rpoB since in some cases it does not resolve the species/subspecies level. We had instances where only the combination of the housekeeping genes with (!) 16SrRNA sequence gave you a correct analysis.

In my opinion, the best strategy to identify bacteria at species or subspecies level is the DNA DNA Hybridizazion with a type strain. However, polyphasic taxonomy that includes morphology,eco- physiology, biochemical and molecular analyses (RISA could be a rapid tool to clusterize at subspecies level see Urz ìet al. (2010), Microbial Ecology, 60:116-129; Pangallo et al., . (2009). International Biodeterioration & Biodegradation, 63: 868-872)

try ITS Sequencing and BLAST.. it works well...

RISA means ribosomal intergenic spacers (ITS) analyses

Polyphasic taxonomy is the best approach as it looks at genetic, phenetic and fatty acid profiles. Though time consuming and relatively expensive this will provide a good insight into the physiological capabilities of the organism being identified.

In my experience also and as suggested by many researchers, polyphasic approach is one of the most reliable choice for bacterial systematics as it is used to discriminate bacterial species based on morphological and biochemical data in addition with information obtained from molecular techniques.

I suggest to use 16S RNA gene-based approach (phylogenetic analysis) and physiological and biochemical properties (Bergy's manuals) determination. Usually this would be fine to determine bacterial species and subspecies.

Armen, while your suggestion may be fine for most of the cases, but in case of the genus Bacillus, it may prove difficult to identify the isolate to sp/subsp. level with 16S and physiological and biochemical properties. We have got an isolate for which we have done FAME, 16S rRNA gene sequence and Biolog (physiological fingerprint based o Burgy's manual). But the strain could not be identified conclusively. It may require further analysis of some molecular markers to ascertain its confirmed identity.

I know this is going to upset some people, but i though i might as well still drop it in here. (looking forward to being proven wrong).

I agree with Pieter that there is no proper definition of species/strain, but i would go just a bit further and say there's no such thing. At lest not to the extent that it provides any extra information. Stumbled over this interesting paper the other day and i think it makes an interesting point.

http://rspb.royalsocietypublishing.org/content/280/1767/20131248.full

Most of the comments specially the last one are helpful but if you're looking for a partially cheep or economic method, as I experienced before, the RFLP is the best. Using one or more of restriction enzyme/s enables you to differentiate most of bacteria in the level of species or subspecies ( with more restriction EZ) .

Dear Kambiz, I too have an environmental bacterial isolate and I want to use RFLP for identification. Is there any RFLP database available to which I can compare my results. If no, then how this could be usefull to me. Kindly expalin a little more. thanks

It completely depends on which bacterial genera you are interested in. Some groups have a higher level of relatedness than others. For example, Bacillus and Lactobacillus species are incredibly difficult to assign using 16S methods alone. It is better practice to use a variety of gene targets (including housekeeping genes, for instance). I agree with some of the earlier comments - assigning isolates to species can be a challenge, since there is almost always debate on how to define a species/strain (for example, some Lactobacillus isolates are constantly being reclassified). Good luck!

I completely agree with the comments of the above scientists for RFLP of the 16S gene and also the ITS region. To my experience you should go for a delta PCR. This will give you the differences between the strains even. Goodluck!

the 16S gene and RFLP etchnique is best method.

@Rajeev: Why do you want to resolve (I took your word) a bacterium to sub species level? As Pieter has said above there are no official rules for designating species and sub species of a bacterium and with 98% similarity two bactria are of same species in case of 16S RNA gene. In the case of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis they have been said to be just one species because of similarity in the sequences of genes.

Just another question: in the case of Bacillus thuringiensis, is it enough to amplifiy any group of cry genes to confirm the specie? Does it need to be confirm ed by 16S rDNA sequencing?

@Maria: Answer to your question is yes one has to amplify cry group genes to confirm B. tthuringiensis along with 16S rDNA sequencing because 16S rDNA sequencing will suggest 100% with Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. And if it is possible you can also go for differential biochemical test given in Bergey's Manual for all the three bacteria.

Other method, mass spectrometry based: MALDI Biotyper from Bruker. Very easy and fast. It is a method that is being implemented in the clinical settings because of its effectiveness. This method is present more and more in the microbiological departments of hospitals. Good luck

Whether you can go down to the species level depends on a number of things. As an example, in the case of the 16S gene, when I look at the v1-v3 region, I can distinguish bifidobacteria perfectly down to the species level. The reason I can do this is because of a number of things. 1: The reference library (SILVA) is very good for bifidobacteria (and for most/many human commensals), if I would for example look at a sample from rats (instead of a sample from humans) I can usually go down to the genus level, and perhaps slightly below this (dividing up a genus into 2 groups or so), but not down to the species level as the reference library is not yet good enough. 2: The length of the 16s region you look at is also important. The longer it is the higher the chance is that you will be able to distinguish species from one another; this directly has to do with point 3: Some groups do not have any meaningfull differences within a particular region of the 16s gene and hence will be indistinguishable if you look at that particular region.

So for choosing a method, it is essential to know on which microbial groups you really want to focus on.

In any case, it would depend on the bacterial group you are working with. Some genus (i.e. Aeromonas) comprise bacterial spcies with 100% homology in their 16S rRNA genes, but with the analysis of housekeeping genes and hybridisation it has been demonstrated that they are different.

I noticed that the answers submitted involved molecular techniques. Unfortunately, the ability to differentiate to species and subspecies level is limited. In this situation, I would suggest reverting to the age-old methods of physiological and biochemical techniques. Of course, these methods will involve the preparation of materials and media and a wide knowledge of the Genus, Species and Subspecies characteristics.

Simple 16S r RNA or 16S r DNA partial sequencing is the best method.

I think that next generation sequencing is the best way.

It depends on the target bacterium. If it belongs to well-studied genus, you can use a number of biochemical, specific PCR markers, even AFLP or rep-PCR in comparison with type strains of all/most of known species with the genus. If your target is in a new species, you better to use MLSA/MLST. If your bacterium has unique 16S and belonged to unknown genus or even higher taxonomic unit, you better sequence complete genome. But, be careful, and prove first that it is a pure culture.

The best is to do polyphasic taxonomic characters likes 16S, Biochemical method, Fatty acid profiling (FAME) and some enzymatic characters are confirms and conclude the species level identification of unknown bacterium.

Its a battery of phenotypic and molecular tests that can be used like other RG members said. Find out from research literatures the most appropriate for your bacteria of interest especially those we call gold standard tests. These vary from one bacterium to the other.

Even a polyphasic approach is not straightforward. The results from 16S, MLSA, FAME etc. can contradict each other. We should progress towards a core genome based analysis. This in combination with an ANI analysis might resolve a reticulated microbial evolution.

I have built up my own software that depends on probability theory. The software uses 15 biochemical testes to identify most commonly isolated medically-important bacteria ( 60 types). I tested it against ATCC strains and was 90% successful.

MLST or similar approaches is quick and reliable for many species, though in some cases will not differentiate well.In this case (especially epidemiological investigations) something like PFGE works for further differentiation, WHole Genome Sequencing and SNP analysis is likely to be the way forward in coming years.

I agree wit Paul Wigley. Our recent experience proved that MLST can give wrong result especially for new group of bacteria.

By using numerical toxanomy methods.

This is not straightforward. The best approach may be a combination of approaches depending on the taxonomic structure and population genetics of the group in question. With some phylogenetic groups, you may have panmictic and clonal taxa needing to be differentiated from each other. Once you understand more of the group in question, the approach may become clearer. For the Epsilonproteobacteria, I think that 16S rRNA gene sequence comparisons, AFLP vs. a good database, whole-cell protein profiling and Multi Locus Sequence Analysis are good places to start. I hasten to add 16S rRNA is not perfect but as long as the caveats are understood it is a good starting point in my view. And sometime well standardised phenotypic assays are useful! If you have a clear idea of particular species, well validated PCR assays can be fast and powerful tools. I have some PCR assay receommendations available on RG I think.

As some answers have indicated polyphasic taxonomy should be used to designate species or subspecies. While the use of various molecular methods have correctly been highlighted, however, there is only minor mention of the phenotypic aspect of polyphasic taxonomy. This highlights a major problem for taxonomy today.

Those who take an interest in taxonomy are largely molecular microbiologists,while proficient traditional microbiologists, that have an active interest in taxonomy, are in decline at an alarming rate. This is not surprising as young researchers are attracted to the latest techniques, which in practice means molecular. This has helped to create a situation whereby papers describing new species and subspecies, as well as changing the genera of established organisms, often passes through the peer review process without adequate scrutiny of phenotype and as a result is unbalanced. Many of those on the research side may be of the opinion that this does not matter, as they pursue the holy grail of a bacterial species concept and classification aligned only to phylogeny. In their world that may be the case, however, the users of taxonomy, are vast in numbers and work in all areas of microbiology. Changes in names and descriptions which contain inadequate phenotype are of no use to them. At a health level, this may have immediate implications for individual patients who are mis-diagnosed with a critical infection and on a broader level may result in delays in the recognition of an emerging or re-emerging disease. It is a major challenge for taxonomy to bring the researchers and diagnosticians together, however, at the moment, I fear that they are succeeding only in moving further apart. Recent evidence is that taxonomy is becoming increasingly a tool for researchers and a cry often heard from diagnosticians is 'why do they have to keep changing the ****** names'.

As someone with an interest in both camps, I find this concerning. So what can be done? I have a few suggestions of my own, but would welcome others from either side of the debate.

Firstly, it should be recognised that microbiologists are a diverse group of workers, covering more areas than most of us could imagine. An effective taxonomy is essential to their work. In this respect, there was recognition previously that taxonomy had to be pragmatic, but I fear that this is rapidly being lost. If sections of the users become disengaged, to my mind, it would represent a failure of the process and do damage to all microbiology.

Secondly,There is a need for the young microbiologists of today to be more aware of microbiology as a broad subject including phenotype.

Finally, with respect to publications of new and re-assigned taxa, several steps could be taken, especially by journal editors and peer reviewers:

1. Ensure that phenotype is rigorously checked at peer review.

2. Ensure that phenotypic comparisons against validated species are made against the phenotype of the species as a whole and not just the type strain, as is happening for an increasing number of publications of novel species.

3. It is recommended that 5 isolates of a novel species are examined for publication of a new species, yet we continually see single species published. Even in the IJSEM, which is the premier journal for taxonomy, this is the case for several papers in each issue. Indeed, it is interesting to note in some cases, how quickly a publication follows on further isolates of the same mew species from the same group of authors, so a little delay could have resulted in a more robust species description.

4. Where a novel organism cannot be differentiated phenotypically from a closely related species, use of genomospecies designation rather than a named species should be considered.

I shall get off my soapbox now, but would make one final sobering comment. A few years ago I was at a meeting on E. coli O157 when one of the delegates, who himself had many publications on the subject, said that there had been a massive amount of money spent on research of O157, a lot of papers written and research careers built and yet we appear to be no further forward in reducing its impact. I would like to extend this statement to taxonomy and would comment that if our taxonomy does not help other microbiologists, a lot of papers will be written and research careers built, but it will be failing a great many of the users.

Hai,

Genus level identification is done by using several biochemical tests, and species level identification is done by using several molecular methods such as 16srRNA ,like that

Identification of bacteria depends in their taxonomic position. Complete 16S sequence will place it into certain genus, MLST will show its relation to known species within this genus, ANI will show possible difference with known species, and if ANI is below 94%, you must complete full study of the bacteria as for new species description: morphology of bacterial cells, physiologic properties by BIOLOG of equal tests, FAME, virulence for pathogenic strains, DNA:DNA hybridization with closest species. Identification of subspecies can relay on a few distinct phenotype traits.

If you're talking sub-species (Salmonella enterica serovars or something), sequencing is likely the only way to go. For species-based differentiation, it highly depends upon how well-characterized your bugs of interest are in the MLST, PFGE, and 16S databases.

You can use Maldi-Tof indentification system. But, please be carreful, it is useful after common cultivation or after by selective media cultivation. Then you have also check normal obligatory phenotypization to confirm Maldi -Tof identification; sometimes you can achieve confusion; that,s why also phenotypization after that is recommended using many disposable identification commercial kits. When you have known species, you can use PCR too to confirm maldi-Tof identification.

DNA-DNA hybridization is the most reliable method. Besides this test is requirement as a proof for a new species.

Yes, I agree with Irene. DNA-DNA hybridization is the most reliable method.

Although DNA-DNA hybridization is now the standard method for new species determination, it will soon be replaced by ANI-analysis. DDH has some major drawbacks which are countered by ANI-analysis (in combination with TETRA-values). Prices of WGS of dropping continually, resulting in cheaper ANI-analysis. Currently, you can sequence 98 genomes for 120€ per genome (>60% coverage, which is enough for ANI).

MALDI-TOF can be used for dereplication, but I would be very careful when applying it for identification, since phylogenetic classification based on MALDI-TOF is far from perfect. Peak profiles need to be a perfect match, one peak difference can mean a different species.

Still, there will always be species that are hard to classify taxonomically (e.g. E. coli) based on DDH or ANI. A polyphasic approach is recommended.