Diagnostic Biomarkers in the Taxonomically Oversized Genus - Clostridium
Author: Vipin Chandra Kalia


Clostridium species are well known for their biotechnological applications: (i) solvents, (ii) biofuels, and (iii) therapeutic and cosmetics, etc. They are among the most dreaded by human beings. Clostridium strains are known to produce deadly toxins responsible for diseases like botulism, tetanus, gas gangrene, pseudomembranous colitis, etc. Cheese industry is one of those, which live in constant fear of getting infected by Clostridium strains. It is important, urgent and imperative to identify the bacteria responsible for causing disease, spoiling food, infecting wounds, before a treatment can be prescribed and implemented.

Diversity of Clostridium

The genus encompasses around 110 species with a G+C content ranging from 24 mol% in Clostridium perfringens at one extreme to 58 mol% in Clostridium barkeri at the other end of the spectrum. Biochemical methods for identifying Clostridium have been holding for quite some time. Things changed dramatically with the discovery of Molecular Biology tools, which revolutionized gene and genome sequencing.

Gene level identification

Bacterial identification has been employing the highly conserved gene: rrs. Individual researchers have been submitting partial and completely sequenced rrs genes to Ribosomal Data Base (RDP database - http://rdp.cme.msu.edu/). The number of rrs gene sequences submitted to RDP is increasing rapidly: RDP Release 11.4 - 3,224,600 seq. The taxonomical classification of Clostridium up to species level using rrs has been running into trouble due to: (i) Very high similarity between two sequences, and (ii) Presence of multiple copies per genome. Consequently, mostClostridium strains have been identified only up to genus level. High heterogeneity in the phylogenetic distribution of strains on the basis ofrrs has been recorded for: C. botulinum, C. perfringens, C. butyricum, C. acetobutylicum, C. beijerinckii, C. novyi, C. kluyveri, etc.

In case rrs based analysis is not proving helpful, scientist generally resort to other genes: (i) Heat shock proteins: hsp65 andhsp70, (ii) ATPase-ß-subunit, (iii) RNA polymerases, (iv) recombinase (recA). In addition, rpoB is used for identifying Mycobacterium, gyrB for Shewanella, Mycobacterium, Pseudomonas, and Acinetobacter, (v) gyrA for defining Bacillus subtilis and related taxa. At times up to 8 genes are needed to be sequenced, if there is no consensus gene specific to a strain.

Genomic tools to decipher novel features

Single gene

Whole genome

Single gene analysis

A novel approach has been used to find out the hidden features of rrs gene sequences of different isolates of Clostridium.

  • Phylogenetic framework
Phylogenetic trees of rrs sequences of 15 Clostridium spp. were analysed to define the limits of the genetic diversity within each of them. Each tree enabled to identify the rrs sequences which could be used as a framework to encompass the organisms belonging to a particular species: C. botulinum, C. perfringens, C. butyricum, C. acetobutylicum, C. beijerinckii, C. novyi, C. kluyveri, C. pasteurianum, C. sporogenes, C. colicanis, C. sardiniense, C. baratii, C. chauvoei, C. subterminale, and C. tetani. A phylogenetic framework of 56 sequences representing 404 rrs sequences of 15 species was used to identify 356 Clostridium strains, which were so far identified only up to the genus i.e., as Clostridium sp.

  • Restriction Endonuclease digestion pattern
In order to validate the authenticity of this Phylogenetic Frame work, all the rrs sequences of the 15 Clostridium spp. were digested with 10 different The unique RE digestion patterns of each species were identified for each of the 15 species.

  • Unique signatures
To further establish that the isolates which have been categorized among known species, 30 nts long unique signatures were detected.

Genome wide analysis

In case of the presence of multiple copies of rrs / genome, one needs to completely sequence all the copies of this gene. However, this exercise does not resolve the problem, because some of the rrs copies from different Clostridium species show 100% similarity. There is thus the risk of mislabeling of strains and overestimation of genetic diversity. Here, whole genome sequence comparisons can help to detect novel marker genes. The strategy employed to find species specific gene features was: (i) to find out genes, common to almost all the genomes and (ii) identify unique RE digestion patterns.

Combinations of genes and their unique RE digestion patterns: recN, dnaJ, secA, mutS, grpE -AluI, BfaI, Tru9I, can help in rapid identification of at least 9 Clostridium species.


  • Hidden features of the rrs gene can be used for bacterial identification.
· Genes common to most organisms within a genus e.g., Clostridium, having unique RE digestion patterns can be employed for diagnostic purposes.


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

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

Kalia VC (2010) Extending genomic limits through metagenomic exploration. J Cosmol 13:3625-3627.

Kalia VC (2013) The Visionary: Prof. Carl R. Woese. Indian J Microbiol 53:245-246. doi: 10.1007/s12088-013-0417-9

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

Kalia VC, Mukherjee T, Bhushan A, Joshi J, Shankar P, Huma N (2011) Analysis of the unexplored features of rrs (16S rDNA) of the genus Clostridium. BMC Genomics 12:18. doi: 10.1186/1471-2164-12-18

Kapley A, Purohit HJ (2009) Genomic tools in bioremediation Indian J Microbiol 49:108-113. doi: 10.1007/s12088-009-0012-2

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

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

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

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

Petrova MI, Lievens E, Malik S, Imholz N, Lebeer S (2015) Lactobacillus species as biomarkers and agents that can promote various aspects of vaginal health. Front Physiol 6:81. doi: 10.3389/fphys.2015.00081

Porwal S, Lal S, Cheema S, Kalia VC (2009) Phylogeny in aid of the present and novel microbial lineages: diversity in Bacillus. PLoS ONE 4:e4438. doi: 10.1371/journal.pone.0004438

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

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 Microiobiol 54:329-336. doi: 10.1007/s12088-014-0452-1

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

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

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:
Researchers in Microbial Biotechnology and Genomics at CSIR-IGIB, Delhi.