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• Bacteria are the most ubiquitous life forms on planet earth, a single gram of soil is said to contain 40 million bacterial cells. Bacteria forms a biomass that exceeds that of plants and animals.

• Because of their abundance most of the bacterial species living on earth have not been identified.

• Various biotechnological techniques have been developed for identifying various bacterial species at the sequence level

• DNA hybridization and 16s rRNA gene sequencing are the most common techniques used for this purpose

Overview of the technique

Isolation of the bacteria

Bacterial DNA extraction

Amplification of the 16s rRNA gene

Sequence a portion of the 16s rRNA gene

Compare the sequenced gene with GenBank to obtain a match

Foremost is the fact that it seems to behave as a molecular chronometer.

The 16s rRNA gene sequence show a high degree of conservation among species. This is assumed to result from the importance of the 16S rRNA as a critical component of cell function.

Few other genes are as highly conserved as the 16S rRNA gene. Although the absolute rate of change in the 16S rRNA gene sequence is not known, it does mark evolutionary distance and relatedness of organisms

16s rRNA is present in almost all bacteria, often existing as a multigene family, or operons.

The 16s rRNA gene (1,500 bp) is large enough for informatics purposes.

16s rRNA gene sequencing studies as opposed to the more cumbersome manipulations involving DNA-DNA hybridization investigations.

16s rRNA gene of various bacteria are extensively studied and is present in many databases

Why use 16s rRNA gene sequencing

Steps Involved in the Process

Isolation of the Bacteria from a sample

• A loopful of the natural sample presumed to contain bacteria is taken and inoculated in a saline solution.

• The inoculated saline solution is serially diluted to reduce the concentration of micro organisms

• After a sufficient dilution has been reached a loopful of inoculum is plated on a sterile nutrient agar plate.

• The plates are incubated at room temperature for 24 hours.

• The colony characteristics are observed to identify the colony of the desired micro-organism

• Different types of media can be used depending on type of micro organism we wish to isolate

• Enrichment media: Media contains substances that stimulates the growth of a desired bacteria and suppresses the growth of unwanted bacteria

(eg: Tetrathionate broth that inhibits coliforms but stimulated bacilli )• Selective Media : Media that allows only a particular type of bacteria

to grow on it . (eg: MacConcey agar for growing gram negative bacteria)

• Differential Media : Media that distinguishes on micro organism type from another growing in the same plate

(eg: Manitol Salt agar , differential for Manitol fermentation)• Anaerobic Media : Media used for growing anaerobic organisms (eg

: Robertson’s cooked meat)

Staining Techniques• After the colony of the desired bacteria

has been identified , the colony is pinched out of the agar plate and stained , for microscopic examination.

• The bacteria can be morphologically identified by simple staining .

• Gram staining is done to check the gram nature of the organism .

• Ziehl-Neelsen stain is used to visualize acid fast bacteria like Mycobacterium tuberculosis and Mycobacterium leprae

• Fluorescent antibody test is used to identify bacteria by the antigens present on their surface

Extraction of DNA from the sample

• A variety of extraction methods can be used for the extraction of DNA for use in 16S rRNA sequencing. The choice of extraction methods rest with the source of the DNA sample and the amount of purity desired.

• For soil bacteria, the most common methods used are bead beating, sonication, enzymatic lysis, etc.

• For bacteria derived from other sources, the mainstream DNA isolation methods like Phenol-Chloroform method, CTAB method are also used.

• Modern laboratories depend on readily available kits to achieve quick, efficient and highly pure DNA Extraction.

Amplification of 16S rRNA Sequence

• The DNA extracted is used as the template for PCR to amplify a segment of about 500 or 1,500 bp of the 16S rRNA gene sequence.

• Broad-based or universal primers complementary to conserved regions are used so that the region can be amplified from any bacteria. The PCR products are purified to remove excess primers and nucleotides.

• The PCR Amplification results in multiple copies of the target DNA Sequence being produced.

• This resulting sequence is then used as the template for the next step of the process known as Cycle Sequencing.

Cycle Sequencing• The next step is a process called cycle sequencing. It is similar to PCR in that it uses DNA

(purified products of the first PCR cycle) as the template. Both the forward and reverse sequences are used as the template in separate reactions in which only the forward or reverse primer is used.

• Cycle sequencing also differs from PCR in that no new template is formed the same template is reused for as many cycles as programmed, usually 25 cycles and the product is a mixture of DNA of various lengths. This is achieved by adding specially labelled bases called dye terminators along with unlabelled bases, which, when they are randomly incorporated in this second cycle, terminate the sequence.

• Thus, fragments of every size are generated. As each of the four added labelled terminator bases has different fluorescent dye, each of which absorbs at a different wavelength, the terminal base of each fragment can be determined by a fluorometer.

• The products are purified to remove unincorporated dye terminators, and the length of each is determined using capillary electrophoresis or gel electrophoresis.

DNA Sequencing

• Since we then know the length and terminal base of each fragment, the sequence of the bases can be determined.

• DNA Sequencing can be done by either the common Sanger Methods, or by using modern DNA Sequencers.

• The two strands of the DNA are sequenced separately, generating both forward and reverse sequences.

• An electropherogram, which is a tracing of the detection of the separated fragments as they elute from the column or are separated in the gel, in which each base is represented by a different colour, can be performed.

• It is possible to have the fragments of various lengths so well separated that every base of a 500-bp sequence can be determined. When ambiguities occur, most of them can be resolved by visual reediting of the electropherogram.

Generating Phylogenetic Trees and Comparing Sequences

• Comparisons are commonly shown as phylogenetic trees and linear alignments.

• A phylogenetic tree is a tree-structured graph used in computational biology to visualize the result of a hierarchical clustering calculation.

• The result of a clustering is presented either as the distance(dissimilarity) or the similarity between the clustered rows or columns depending on the selected distance measure.

• Methods commonly used for generating phylogenetic trees are:

NJ method (Neighboor-joining),

UPGMA method (Unweighted pair group method with arithmetic averages)

WPGMA method (Weighted pair group method with arithmetic averages)

• The methods are comparable, and the major groupings are preserved if the isolates are closely related.

• However, when the taxa being compared are less closely related, the phylogenetic tree relationships are more strongly affected by the program used for accurate 16S rRNA gene sequence identification of organisms.

• In such cases, we are dependent on accurate sequences in databases, appropriate names associated with those sequences, and an accurate sequence for the isolate to be identified.

• There are several reasons why sequence databases can vary and may not accurately link a name with a sequence and, further, with a correct relative placement of the sequence among other bacterial sequences. It could be that the type strain or strains in certified collections such as ATCC were incorrectly named or classified by biochemical means or that the descriptions are just wrong.

• A familiar example of an organism being placed in the wrong genus is “Corynebacterium aquaticum.” Although “C. aquaticum” has, superficially, the same morphology as the corynebacteria, it is genetically distant from the genus Corynebacterium and is more closely related to organisms in the genus Microbacterium.

• When examined by genotypic methods, the ATCC strains previously deposited as Corynebacterium xerosis were found to be diverse and incorrectly identified.

• There is also the misplacement of well-defined species presumably within a single genus but actually found in many taxonomic groups. Species of the genus Enterobacter are found associated with five different genera. Enterobacter is a good example of what is called a polyphyletic genus, as is Citrobacter

• A third reason for error applies primarily to unverified databases such as GenBank, which accept any linked name and sequence that is sent to them.

• It was this sort of consideration that propelled the development of the proprietary MicroSeq database of verified type strains and the RIDOM database.

APPLICATIONS OF BACTERIAL IDENTIFICATION USING 16S rRNA SEQUENCING

1. Identifying unidentified bacteria or isolates with ambiguous profiles.

• One of the most attractive potential uses of 16S rRNA gene sequence informatics is to provide genus and species identification for isolates that do not fit any recognized biochemical profiles, for strains generating only a “low likelihood” or “acceptable” identification according to commercial systems, or for taxa that are rarely associated with human infectious diseases.

• The cumulative results from a limited number of studies to date suggest that 16S rRNA gene sequencing provides genus identification in most cases (>90%) but less so with regard to species (65 to 83%), with from 1 to 14% of the isolates remaining unidentified after testing.

• Difficulties encountered in obtaining a genus and species identification include the recognition of novel taxa, too few sequences deposited in nucleotide databases, species sharing similar and/or identical 16S rRNA sequences, or nomenclature problems arising from multiple genomovars assigned to single species or complexes.

• Criteria for species identification

Minimum: >99% sequence similarity; ideal: >99.5% sequence similarity

Sequence match is to type strain or reference strain of species that has undergone DNA-relatedness studies

For matches with distance scores <0.5% to the next closest species, other properties, including phenotype, should be considered in final species identification

2. Identification of Mycobacteria

• Mycobacteria are in general slow-growing and difficult to identify. Thus, they were an important group of organisms in early important studies establishing the usefulness of 16S rRNA gene sequencing for clinical microbiology.

• More recently, there have been several additional studies comparing the identification of mycobacteria by 16S rRNA gene sequence and phenotypic methods. In all of the studies, the accuracy of 16S rRNA gene sequencing in the identification to the species level was judged to be superior overall to phenotypic methods.

• Overall, by providing for the accurate identification of species in the database and the taxonomic placement if not complete identification of novel species, 16S rRNA gene sequence analysis of mycobacteria seems to be the most accurate method available.

3. Discovery and Description of Novel Pathogens

• In clinical microbiology practice, novel organisms are generally first recognized by an aberrant phenotype or niche.

• If these observations are followed by 16S rRNA gene sequencing, the sequence often indicates that the organism is only an unusual phenotype of a known taxon. However, at this time, perhaps 10 to 20% of the isolates might not match any other described organism and thus might be a novel organism and an even higher percentage might be previously described organisms but not strains usually encountered in clinical practice.

• A medium to large clinical microbiology laboratory can be expected to isolate a few novel organisms per month.

Conclusion

• The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods.

• Comparison of the bacterial 16S rRNA gene sequence has emerged as a preferred genetic technique. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, can be routinely used for identification of mycobacteria, and can lead to the recognition of novel pathogens and noncultured bacteria.

• Problems remain in that the sequences in some databases are not accurate, there is no consensus quantitative definition of genus or species based on 16S rRNA gene sequence data,

Conclusion

• The proliferation of species names based on minimal genetic and phenotypic differences raises communication difficulties, and micro heterogeneity in 16S rRNA gene sequence within a species is common.

• Despite its accuracy, 16S rRNA gene sequence analysis lacks widespread use beyond the large and reference laboratories because of technical and cost considerations. Thus, a future challenge is to translate information from 16S rRNA gene sequencing into convenient biochemical testing schemes, making the accuracy of the genotypic identification available to the smaller and routine clinical microbiology laboratories.

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