API 20E identification strips were previously inoculated with 6 different isolates of bacteria
For morphological and also for biochemical tests, bacteria are routinely identified. The identification could have been done based on its phenotypic analysis and after that by comparing it with the known trait of earlier strains. Then again, it consumes too much time and is also labor intensive. To solve this issue different companies, present in the market have prepared and offered much more useful alternatives to this which save time and are also less labor intensive, and this has resulted in simplifying the overall process. And one of the best examples of it is API 20E strips for the tests, and in particular, it has been developed by Biomerieux. In this practical, we have used API 20E strips to determine the six unknown isolates and identified what they are (AL-Hadrawi, et al., 2019).
API 20E is the plastic strip that contains only 20 chambers for test and this all is in small size it also contains dehydrated media along with the chemically defined compositions for each one of the chamber. Usually they are being used to detect the activity of the enzyme, and if talk more accurately used in related to the fermentation of carbohydrates of the catabolism of proteins or even for amino acids by the inoculated organism. It is used for the identification of gram-positive and negative bacteria and also yeast, it has been observed based on the different tests that it is capable of delivering accurate identification based on extensive databases. The full form of API is an analytical profile index it is a panel for the identification of biochemicals (Gruzdys, V., et al 2020). The objective of this particular practical experiment is to learn about the use of API 20E by performing the required procedure and step and getting the desired result.
To identify and differentiate between the given members of the family Enterobacteriaceae
- API 20E identification strips were previously inoculated with 6 different isolates of bacteria.
- 16S rRNA sequence data.
1 to confirm the culture and to test, a quick oxidase test for cytochrome c oxidase has been performed.
2 we picked one of the slides among the 6 different present slides and made a suspension of distilled water which was sterilized too.
3 after that we have taken the API20E strip and then put the reagent into the 20 separate compartments which are present in the strip after that we used the Pasteur pipette, and filled it up with the suspension of bacteria.
4 Abd than after it we put the oil for sterile purposes into the ADH, LDC, ODC, H2S, and URE blocks.
5 we then added some of the water drops into the tray and added the API test strip and then just closed the tray.
6 after that we marked the tray with the number for the identification purpose so that we can remember which was it and don’t mix things up for most of the different things we put dates and initials to for more accuracy.
7 after all of that we incubate the tray at a constant temperature of 37 degrees Celsius about in between 18 to 24 hours and after that, they can provide the right desired result and are ready to observe (Dilnessa, et al., 2016).
(6 of the strips were inoculated, which are as per instructed by the manufacturers, and all of the six strips with a suspension of one of six different bacteria were incubated overnight at 37 degrees Celsius and in a humid chamber.
The dehydrated substrates present on the strips detect any kind of activity performed by the enzymes and it required a suitable environment to do so otherwise experiment will fails)
The result is based on the change that it exhibits after the experiment in terms of color, odor, form, elevation, margin, etc.
For example, different interpretations may be observed or used the determination the result.
For some of the cells in the tray, the color change will be the indicator and it is the first visible indicator just after the result. But before making any sort of interpretation some of the reagents has to be put into it. The reagent that will be used are TDA, and IND VP, this will require in very minor quantities.
After that mark the API reading scale which is a color chart based on positive and negative on the lid of the tray. And after that based on the result, the seven-digit score will be allocated after adding the value which will be shown in all three individuals and it will provide the 7-digit number.
The shape could be changed.
- form could be (punctiform, circular, filamentous, irregular, rhizoid, or spindle)
- Elevations are as (flat, raised, convex, pulvinate, umbonate)
- margin is as (entire, undulate, lobate, erose, filamentous, curled)
Based on the result that the experiment shows based on the above-mentioned criteria the outcome which has come for all the six experiments are as follows (Temitope, et alj.,2016).
The results of the experiments are as follows
RESULTS BASED ON OBSERVATION The meaning of the above-used indicators are mentioned here
W is for white, Y is for yellow, o is for orange, PL is for pale yellow, BL is for blue, LP is for light pink, p is for pink, Br is for brown, B is for black, G is for green, LB is for light brown.
A – klebsiella pneumoniae
B – pseudomonas flurescens
C – Escherichia coli
D – Enterobacter colacae
E – proteus mirabilis
F – Serratia marcesens (Aruwa, & Olatope, 2015).
On the behalf of the result that has been shown in the result, it became easy to identify what the substance is based on the color that has it shown after missing reagents of different types concluding the color, result, and the value we get our seven digits numerical value, and that results in providing us the name of the substance and hence the practical was done and the result was concluded with using API 20 E. and it has been a day taking practical as because of the time taken by the reagents to work.
Analysis of 16s rRNA sequencing data
In the name 16s rRNA, 16s basically stands for ribosomal ribonucleic acid, and in this S is for the Svedberg it is basically a unit for the purpose of measurement. And the most important part of the small subunit of prokaryotic ribosomes is rRNA also for the mitochondria and also for the chloroplasts. Generally many get confused with protein-coding gene is 16s RNA but it, not the ribosome is a must for a binding site which makes it able to produce the proteins. In comparison to the traditional method for sequencing, it has been finding out that the technique of sequencing which used 16s rRNA is much more efficient in both terms whether its financial reason or in reducing the manual handling not just fast but cost-effective and that’s why it is being the choice of the microbiologist and also for commercial purpose. It does have its issues too but has many positive results in comparison to negative ones which makes it much more reliable but not suitable for each and every situation. It is the central structure of a component in bacterial and archeal 30S ribosomal subunit and more than that it is essential for the synthesis of the protein and also acts as the stabilization of codon and anticodon pairing at the A site in the ribosome during mRNA translation.
This experiment is about the result of DNA sequencing using 16s rRNA, it refers to the accurate arrangement of nucleotides, or bases, in a molecule or more specifically in a DNA molecule in the casual laboratory technique for knowing arrangement. Its been for decades now that 16s rRNA has been there and used for sequence-based bacterial analysis. And sequencing of full genes has been possible recently. It has been seen that it has almost 1500 base pairs which is an approximate value but not less than 1500, and this is also from conserved reason and that results in brad taxonomic spectrum, and it is having hypervariable region from the v1 to the v9 and that again leads to the taxonomic level increase.
There is a complexity of DNA-DNA hybridization and because of it 16s rRNA sequencing has been used for identifying bacteria at the species level it also helps in assisting with differentiating between bacterial species which are very closely related (Garrido-Cardenas, et al., 2017)
It has been observed that many clinical laboratories is being relying on this method to identify unknown pathogenic strains. In an analysis of sequenced bacterial genomes in silico, there has been a total number of more than 7 k copies of 16s rRNA has been identified and which is having almost an average of 4 copies per genome. And more than that around 15 % of genomes is having a single print of 16s rRNA, almost 21 % is having two copies of it, and 3 to 7 copies on every genome is being found.
The nine variable regions are being interpreted by the highly conserved 16s sequence from the 1500 bp 16s rRNA gene. Sanger sequencing is the region of successful sequencing of the entire gene. Some of the basic things that require are such as cloning genes, generating, and assembling approx. two to three reads for every clone, which will produce the limited sampling at a very high cost and also at very high efforts too.
It helps in speeding the laboratory process for diagnosis of infections very fast. It shortens the long time gap it usually takes to know the slow-growing bacteria and increases the overall clinical diagnosis and guides prompt antibiotic treatment.
16s rRNA Sequencing
In today’s time, rDNA sequence analysis or 16S rRNA is becoming the major tool of choice in terms of it helps in knowing the relation between bacteria, and it is used widely for identification. All the bacteria which are present out there is having 16s rRNA gene in them and not exactly the same but the somewhat similar form is present in all cells which also include the eukaryotic cell.
16s ribosomal databases are EzBioCloud, ribosomal database project, SILVA, and Green Genes.
Ezbiocloud database also known as ExTaxon has a total hierarchical taxonomic system and it has more than 60 k bacteria and archaea species variety and it is having valid data of bacteria of about 15 k which has been had published names.
A ribosomal data project is a kind of program which has data on bacteria along with the related services and programs regarding them. This data can be available by any e-mail server. And it is also used as a bioinformatic tool development because of the huge size it has that is for creating curated databases (Heerema, et al., 2016).
Silva works as a quality check provider and has updated datasets of aligned small or large subunit ribosomal RNA sequences and this is all for all three domains of life Abellan-(Schneyder, et al., 2021).
Green genes is also a type of quality-controlled, comprehensive 16s rRNA gene database for the references, and has a de novo hylogeny-based taxonomy. And one must note that it utilizes taxonomic terms which were proposed by phylogenetic methods years ago.
PCR and NGS applications – 16s rRNA contains hypervariable regions and it has the property of providing the signature of specific sequences it has been found that it is very useful in terms of identifying bacteria. And it results in the prevalence in medical biology as a quick and less costly option for the different phenotypic methods of bacterial identification (Church, et al., 2020)
Bacterial identification using 16s rRNA sequencing
Identification of that genus and species which has not fit and recognized any biochemical profile, for strains and which are generating low-level identification as per the commercial system, using 16s rRNA technique for gene sequencing is been seen as the most attractive potential usable technique because of its large database and cost-effectiveness over the other different method which also involves too much human need but this technique, not just the time but also the manpower, and that makes it more suitable for commercial purposes in comparison to any other kind of technique that is present in the current time.
It has been observed that for the case of genus identification the data which is being provided by using this technique is almost more than 90 % accurate and even more than that but not less on the other hand if we talk about species it has an accuracy level of nearly in between 65 to 83 %. (Yaish, et al., 2016).
The reason why it is highly conserved is that the 16s rRNA genes have been found in each and every prokaryotic cells, and all bacterias have it because it is not possible for an organism to translate mRNA without having 16s rRNA and it is a part of small subunit in ribosome which means all bacteria have it. The genes are essential that’s why and very highly conserved.
After analyzing it in different scenarios and places it has been clear that gene sequence information which was presented as the result of using the technique 16s rRNA has provided accurate data only for the bacteria in public health or clinical settings, but on other hand it has also been observed that the data which came out after the using this technique for species is less accurate overall the result shows that after observing each and every aspect of data is that it is not all that bullet proof or fool proof and not applicable to each and every scenario, it indeed has it some pros which give this technique an upper hand on most of the scenarios but that doesn’t fit for every possible scenario. And this is came out as the major issue with this technique (Clarridge III, 2004).
Abellan-Schneyder, I., Matchado, M.S., Reitmeier, S., Sommer, A., Sewald, Z., Baumbach, J., List, M. and Neuhaus, K., 2021. Primer, pipelines, parameters: issues in 16S rRNA gene sequencing. msphere, 6(1), pp.e01202-20.
AL-Hadrawi, H.A.N., AL-Harmoosh, R.A. and AL-Fatlawy, H.N.K., 2019. Detection of Some Virulence Factors of Clinical V. cholerae isolates in Najaf/Iraq. Journal of Pharmaceutical Sciences and Research, 11(2), pp.375-379.
Aruwa, C.E. and Olatope, S., 2015. Characterization of Bacillus species from convenience foods with conventional and API kit method: A comparative analysis. J Appl Life Sci Int, 3(1), pp.42-48.
Church, D.L., Cerutti, L., Gürtler, A., Griener, T., Zelazny, A. and Emler, S., 2020. Performance and application of 16S rRNA gene cycle sequencing for routine identification of bacteria in the clinical microbiology laboratory. Clinical Microbiology Reviews, 33(4), pp.e00053-19.
Clarridge III, J.E., 2004. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clinical microbiology reviews, 17(4), pp.840-862.
Corless, C.E., Guiver, M., Borrow, R., Edwards-Jones, V., Kaczmarski, E.B. and Fox, A.J., 2000. Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. Journal of clinical microbiology, 38(5), pp.1747-1752.
Dilnessa, T., Mengistu, G.D.G. and Bitew, A., 2016. Journal of Medical Microbiology & Diagnosis.
Garrido-Cardenas, J.A., Garcia-Maroto, F., Alvarez-Bermejo, J.A. and Manzano-Agugliaro, F., 2017. DNA sequencing sensors: an overview. Sensors, 17(3), p.588.
Gruzdys, V., Signorelli, H. and Johnson-Davis, K.L., 2020. NicAlert™ test strip performance comparison with LC-MS/MS and immunoassay methods for nicotine and cotinine. Archives of Clinical Toxicology, 2(2), pp.19-24.
Heerema, S.J. and Dekker, C., 2016. Graphene nanodevices for DNA sequencing. Nature nanotechnology, 11(2), pp.127-136.
Kim, S.H., Ganji, M., Kim, E., van der Torre, J., Abbondanzieri, E. and Dekker, C., 2018. DNA sequence encodes the position of DNA supercoils. Elife, 7, p.e36557.
Temitope, O.O., Fasusi, A.O., Ogunmodede, A.F. and MI, E., 2016. ANTIBACTERIAL SENSITIVITY PROFILE, ISOLATION AND IDENTIFICATI ON OF BACTERIA USING API 20E KITS FROM HOSPITAL SOLID WASTE DUMP SITES OF IKARE-AKOKO, STATE SPECIALIST HOSPITAL, ONDO STATE, NIGERIA.
Yaish, M.W., Al-Harrasi, I., Alansari, A.S., Al-Yahyai, R. and Glick, B.R., 2016. The use of high throughput DNA sequence analysis to assess the endophytic microbiome of date palm roots grown under different levels of salt stress. Int. Microbiol, 19(3), pp.143-155.