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Searching the Dreadful Yersinia in a Crowd

BY: Vipin Chandra Kalia | Category: Environmental-Biotechnology | Submitted: 2016-10-31 22:09:41
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Article Summary: "Yersinia infection is lethal to human beings. It is one of the most dreadful pathogens. The need is to identify them and initiate the treatment before it is too late. Culture techniques are the best but not rapid. Molecular techniques based on 16S rDNA (rrs) gene analysis are limited by the presence of 6-7 copies of this gene/ge.."


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Searching the Dreadful Yersinia in a Crowd
Author: Vipin Chandra Kalia

Introduction

Yersinia is one of the most dreaded pathogens. The three most important Yersinia species which infect human being are: Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis. Y. pestis causes plague – The Black Death. The host can die within 24 h of contracting the disease. It spreads from rodents to mammals – humans, dogs and cats. An unusual number of deaths among rodents is an indication of an outbreak of plague. Once the host dies, the fleas bite and infect mammals. The infection is easy to spread. If it goes unchecked for some time, it could be fatal and a public health hazard. The other two species: Y. enterocolitica and Y. pseudotuberculosis cause a diarrheal illness called as yersiniosis,

Diagnosis and Treatment

The process of diagnosis and treatment can be initiated only after the symptoms start appearing. Conventionally, pus from the swollen lymph node/ blood/ sputum can be cultured. This is a slow process and takes a few days. The need is to test it rapidly. Biochemical assays are based on the presence of specific plague antigen. Y. pestis antibodies in a blood sample can be detected within a few hours. However, the assay is performed in a specialized laboratory under bio-safety conditions. Y. enterocolitica and Y. pseudotuberculosis can be easily distinguished through biochemical assays.

Culture Techniques

In spite of extensive and rapid progress being made in detecting pathogenic organisms, the culturing technique is still regarded as the best. These methods are limited by their long incubation periods. In contrast, systems like BIOLOG and API 20E are commercially available but are based on costly consumables and equipments. Hence, their use for routine purposes is not feasible.

Immunological techniques

Immunoassays detect and identify microbes without the need to culture them. Latex agglutination assay employs latex beads coated with antibodies specific to a pathogen, which agglutinates antigens. The resultant precipitate can be easily viewed. In Immunomagnetic separation (IMS) assay, the antibodies fish out the organism of interest. Subsequently, it is verified using PCR or ELIZA. Other immunological assays use swine antibodies against lipopolysaccharide, which detects the O-antigens. A more precise assay is immunosensor based on the surface plasmon resonance, which can detect bacteria even at low cell density. It has been effective in case of Y. enterocolitica infected food products but every assay need specific antibodies.

Molecular techniques

Assays based on the use of genetic material (DNA) have proved to be more rapid, precise and economical. These assays include: PCR, microarray, colony hybridization and loop-mediated isothermal amplification (LAMP) along with Restriction Endonuclease digestion (RE).

16S rRNA (rrs), the conserved gene is used extensively to identify (https://rdp.cme.msu.edu/). However, under two scenarios, this gene is not effective: (i) very closely related taxa, and (ii) presence of multiple copies of this gene in each genome. The process becomes complicated and uneconomical. An obvious option is to use other conserved genes, such as: recA, gyrA, gyrB, rpoB, etc. The genes routinely used to identify Yersinia, include: ail, inv, yst, myf, yop, vir, etc.


Other assays employed for identifying Yersinia are: Microarray, Loop-mediated isothermal amplification (LAMP), a silicon based optical biosensor, MALDI-TOF mass spectrometry, and FTIR spectroscopy. These techniques are effective for high-throughput works.

Searching universal biomarkers for Yersinia

Yersinia spp. contain 6-7 copies of the gene rrs in each genome. They show very high similarity among themselves. To circumvent this problem, it becomes imperative to use other genes. It was realized that although many genes are being used but there is no consensus among various researchers. In addition, it was also revealed that genes being studies may not be present in all the species and strains of Yersinia: ail, inv, yst, myf, vir, and yop. These genes cannot be used as biomarkers for this genus.

An extensive analysis of 51 completely sequenced genomes belonging to 10 species of Yersinia was carried out to search genes common to them. Of the 3219-5596 genes carried by each genome, 304 genes were to them. Restriction Endonucleases (REs) digestion of 34 out of 304 genes with AluI, BfaI, BfuCI, RsaI, CviAII, TaqI, HpyCH4V, Tru9I, Hin1I, and HaeI, revealed unique combinations of genes and REs. The following genes-RE combinations can be used as biomarkers for identifying quite a few Yersinia strains with high precision: carB, gltX, malE, rlmL, fadJ, gluM, ileS, nusA, and ribD,

Unique selling points

· The RE digestion fragments were easily distinguished on the basis of their size and number.

· The genes were common and present in all the strains.

· The genes can be used for diagnostic purposes.

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About Author / Additional Info:
Researcher in Microbial Biotechnology and Genomics at CSIR-IGIB, Delhi.

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