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.

References

1. Agarwala M, Choudhury B, Yadav RNS (2014) Comparative study of antibiofilm activity of copper oxide and iron oxide nanoparticles against multidrug resistant biofilm forming uropathogens. Indian J Microbiol 54:365–368. doi:10.1007/s12088-014-0462-z

2. Alipiah NM, Shamsudin MN, Yusoff FM, Arshad A (2015) Membrane biosynthesis gene disruption in methicillin-resistant Staphylococcus aureus (MRSA) as potential mechanism for reducing antibiotic resistance. Indian J Microbiol 54:41-49. doi:10.1007/s12088-014-0488-2

3. Ayyadurai S, Flaudrops C, Raoult D, Drancourt M (2010) Rapid identification and typing of Yersinia pestis and other Yersinia species by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry. BMC Microbiol 10:285. doi:10.1186/1471-2180/10/285

4. Bhagat N, Virdi JS (2007) Distribution of virulence associated genes in Yersinia enterocolitica biovar 1A correlates with clonal groups and not the source of isolation. FEMS Microbiol Lett 266:177–183. doi:10.1111/j.1574-6968.2006.00524.x

5. Bhushan A, Joshi J, Shankar P, Kushwah J, Raju SC, Purohit HJ, Kalia VC (2013) Development of genomic tools for the identification of certain Pseudomonas up to species level. Indian J Microbiol 53:253–263. doi: 10.1007/s12088-013-0412-1

6. Bhushan A, Mukherjee T, Joshi J, Shankar P, Kalia VC (2015) Insights into the origin of Clostridium botulinum strains: evolution of distinct restriction endonuclease sites in rrs (16S rRNA gene). Indian J Microbiol 55:140–150. doi: 10.1007/s12088-015-0514-z

7. Gupta, V, Gulati P, Bhagat N, Dhar MS, Virdi JS (2015) Detection of Yersinia enterocolitica in food: an overview. Eur J Clin Microbiol Infect Dis 34:641-650.doi:10.1007/s10096-014-2276-7

8. Kalia VC (2015) Let's explore the latent features of genes to identify bacteria. J Mol Genet Med 9:e105. doi:10.4172/1747-0862.1000E105

9. Kalia VC, Kumar P (2015) Genome wide search for biomarkers to diagnose Yersinia infections. Indian J Microbiol 55:366-374. doi:10.1007/s12088-015-0552-6

10. Kalia VC, Kumar P, Kumar R, Mishra A, Koul S (2015) Genome wide analysis for rapid identification of Vibrio species. Indian J Microbiol 55:375-383. doi:10.1007/s12088-015-0553-5

11. Kekre A, Bhushan A, Kumar P, Kalia VC (2015) Genome wide analysis for searching novel markers to rapidly identify Clostridium strains. Indian J Microbiol 55:250257.doi: 10.1007/s12088-015-0535-7

12. Kingston JJ, Radhika M, Roshini PT. Raksha MA, Murali HS, Batra HV (2010) Molecular characterization of lactic acid bacteria recovered from natural fermentation of beet root and carrot Kanji. Indian J Microbiol 50:292–298. doi: 10.1007/s12088-010-0022-0

13. Koul S, Kalia VC (2016) Comparative genomics reveals biomarkers to identify Lactobacillus species. Indian J Microbiol 56:253–263. doi:10.1007/s12088-016-0605-5

14. Koul S, Kumar P, Kalia VC (2015) A unique genome wide approach to search novel markers for rapid identification of bacterial pathogens. J Mol Genet Med 9:194. doi: 10.4172/1747-0862.1000194

15. Kumar R, Koul S, Kumar P, Kalia VC (2016) Searching biomarkers in the sequenced genomes of Staphylococcus for their rapid identification. Indian J Microbiol 56:64-71.doi:10.1007/s12088-016-0565-9

16. Liu Q, Wang S, Zhi J-F, Ming H, Teng D (2013) Efficient production of lactic acid from sweet sorghum juice by a newly isolated Lactobacillus salivarius CGMCC 7.75. Indian J Microbiol 53:332-336. doi: 10.1007/s12088-013-0377-0 17. Mahale KN, Paranjape PS, Marathe NP, Dhotre DP, Chowdhury S, Shetty SA, Sharma A, Sharma K, Tuteja U, Batra HV, Shouche YS (2014) Draft genome sequences of Yersinia pestis strains from the 1994 plague epidemic of Surat and 2002 Shimla outbreak in India. Indian J Microbiol 54:480-482. doi: 10.1007/s12088-014-0475-7 18. Moroeanu VI, Vamanu E, Paun G, Neagu E, Ungureanu OR, Eremia SAV, Radu GL, Ionescu R, Pelinescu DR (2015) Probiotic strains influence on infant microbiota in the in vitro colonic fermentation model GIS1. Indian J Microbiol 55:423-429. doi: 10.1007/s12088-015-0542-8

19. Prakash O, Pandey PK, Kulkarni GJ, Mahale KN, Shouche YS (2014) Technicalities and glitches of terminal restriction fragment length polymorphism (T-RFLP). Indian J Microiobiol 54:255-261. doi:10.1007/s12088-014-0461-0

20. Prakasham RS, Kumar BS, Kumar YS, Kumar KP (2014) Production and characterization of protein encapsulated silver nanoparticles by marine isolate Streptomyces parvulus SSNP11. Indian J Microbiol 54:329-336. doi: 10.1007/s12088-014-0452-1

21. Saxena A, Mukherjee M, Kumari R, Singh P, Lal R (2014) Synthetic biology in action: Developing a drug against MDR-TB. Indian J Microbiol 54:369-375. doi: 10.1007/s12088-014-0498-0

22. Shang Z, Wang H, Zhou S, Chu W (2014) Characterization of N-acyl-homoserine lactones (AHLs)-deficient clinical isolates of Pseudomonas aeruginosa. Indian J Microbiol 54:158-162. doi:10.1007/s12088-014-0449-9

23. Wang R, Fang S, Xiang S, Ling S, Yuan J, Wang S (2014) Generation and characterization of a scFv antibody against T3SS needle of Vibrio parahaemolyticus. Indian J Microbiol 54:143-150. doi: 10.1007/s12088-013-0428-6

24. Yu S, Peng Y, Chen W, Deng Y, Zheng Y (2014) Comparative genomic analysis of two-component signal transduction systems in probiotic Lactobacillus casei. Indian J Microbiol 54:293-301. doi: 10.1007/s12088-014-0456-x

25. Yu S, Peng Y, Zheng Y, Chen W (2015) Comparative genome analysis of Lactobacillus casei: Insights into genomic diversification for niche expansion. Indian J Microbiol 55:102-107. doi: 10.1007/s12088-014-0496-2



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