ABSTRACT Aeromonas hydrophila is an important bacterial pathogen which causes the hemorrhagic septicemia in fishes, amphibians and humans. Genetic relationships of diverse isolates of A. hydrophila were recovered from fish and water sources. These isolates were investigated by flanked region of 16S and 23S Ribosomal DNA (rDNA) internal transcribed spacer (ITS). Here we analyzed polymorphism of PCR-amplified 16S-23S rDNA ITS and their revealed band pattern consisting of one to four DNA fragments. The fragment size ranged from 500 to 1000 bp. The DNA band patterns revealed a considerable genetic diversity in interaspecies. The 750 bp size of band was common in all isolates of A. hydrophila except one Isolates AH21. The tRNA-Glu sequences were identified from 750 bp size of ITS region that could be used as strain level potential genotypic markers.
Cite this paper
V. Singh, I. Mani and D. Chaudhary, "Molecular Assessment of 16S-23S rDNA Internal Transcribed Spacer Length Polymorphism of Aeromonas hydrophila," Advances in Microbiology, Vol. 2 No. 2, 2012, pp. 72-78. doi: 10.4236/aim.2012.22009.
 R. P. Kokka, J. M. Janda, L. S. Oshiro, M. Altwegg, T. Shimada, R. Sakazaki and D. J. Brenner, “Biochemical and Genetic Characterization of Autoagglutinating Phenotypes of Aeromonas Species Associated with Invasive and Noninvasive Disease,” Journal of Infectious Disease, Vol. 163, No. 4, 1991, pp. 890-894.
 M. Dorsch, N. J. Ashbolt, P. T. Cox and A. E. Goodman, “Rapid Identification of Aeromonas Species Using 16S rDNA Targeted Oligonucleotide Primers: A Molecular Approach Based on Screening of Environmental Isolates,” Journal of Applied Bacteriology, Vol. 77, No. 6, 1994, pp. 722-726. doi:10.1111/j.1365-2672.1994.tb02825.x
 D. Qian. Y. Chen, J. Shen and Z. Shen, “Serogroups, Virulence and Hemolytic Activity of Aeromonas hydrophila Which Caused Fish Bacterial Septicaemia,” Wei Sheng Wu Xue Bao, Vol. 35, No. 6, 1995, pp. 460-464.
 A. S. Yadav and A. Kumar, “Prevalence of Enterotoxigenic Motile Aeromonads in Children, Fish, Milk and Ice-Cream and Their Public Health Significance,” Southeast Asian Journal of Tropical Medical and Public Health, Vol. 31, Suppl. 1, 2000, pp. 153-156.
 H. Saeki, N. Matsuda, T. Tamura, N. Masuda and A. Yonei, “A Case of Severe Septicemia Due to Aeromonas hydrophila,” Masui, Vol. 51, No. 2, 2002, pp. 193-195.
 V. Minnaganti, P. Patet, D. Iancu, P. Schoch and B. Cunha, “Necrotizing Fasciitis Caused by Aeromonas hydrophila,” Heart and Lung, Vol. 29, No. 4, 000, pp. 306-308.
 B. Austin and D. A. Austin, “Bacterial Fish Pathogens: Diseases in Farmed and Wild Fish,” Praxis Publishing, Chichester, 1999.
 J. M. Janda, S. L. Abbott, W. K. Cheung and D. F. Hanson, “Biochemical Identification of Citrobacteria in the Clinical Laboratory,” Journal of Clinical Microbiology, Vol. 32, No. 8, 1994, pp. 1850-1854.
 T. R. Karl, R. W. Knight and N. Plummer, “Trends in High-Frequency Climate Variability in the Twentieth Century,” Nature, Vol. 377, No. 6546, 1995, pp. 217-220.
 R. M. Carr, U. J. Blumenthal and D. D. Mara, “Guidelines for the Safe Use of Wastewater in Agriculture: Revisiting WHO Guidelines,” Water Science and Technology, Vol. 50, No. 2, 2004, pp. 31-38.
 A. Kozinska, “Dominant Pathogenic Species of Mesophilic Aeromonads Isolated from Diseased and Healthy Fish Cultured in Poland,” Journal of Fish Disease, Vol. 30, No. 5, 2007, pp. 293-301.
 M. Tacao, A. Alves, M. J. Saavedra and A. Correia, “BOX- PCR Is an Adequate Tool for Typing Aeromonas spp.,” Antonie van Leeuwenhoek, Vol. 88, No. 2, 2005, pp. 173-179. doi:10.1007/s10482-005-3450-9
 M. C. Rodriguez-Barradas, R. J. Hamill, E. D. Houston, P. R. Georghiou, J. E. Clarridge, R. L. Regneryn and J. E. Koehler, “Genomic Fingerprinting of Bartonella Species by Repetitive Element PCR for Distinguishing Species and Isolates,” Journal of Clinical Microbiology, Vol. 33, No. 5, 1995, pp. 1089-1093.
 L. Soler, M. J. Figueras, M. R. Chacón, J. Guarro and A. J. Martinez-Murcia, “Comparison of Three Molecular Methods for Typing Aeromonas popoffii Isolates,” Antonie Van Leeuwenhoek, Vol. 83, No. 4, 2003, pp. 341-349.
 B. Vold, “Structure and Organization of Genes for Transfer Ribonucleic Acid in Bacillus subtilis,” Microbiology Review, Vol. 49, No. 1, 1985, pp. 71-80.
 J. Welsh and M. McClelland, “Genomic Fingerprints Produced by PCR with Consensus tRNA Gene Primers,” Nucleic Acids Research, Vol. 19, No. 4, 1991, pp. 861-866.
 S. Jinks-Robertson and M. Nomura, “Ribosomes and tRNA,” In: F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter and H. E. Umbarger, Eds., Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, American Society for Microbiology, Washington DC, 1987, pp. 1358-1385.
 M. McClelland, C. Petersen and J. Welsh, “Length Polymorphism in tRNA Intergenic Spacers Detected by Using the Polymerase Chain Reaction Can Distinguish Streptococcal Strains and Species,” Journal of Clinical Microbiology, Vol. 30, No. 6, 1992, pp. 1499-1504.
 V. Singh, G. Rathore, D. Kapoor, B. N. Mishra and W. S. Lakra, “Detection of Aerolysin Gene in Aeromonas hydrophila Isolated from Fish and Pond Water,” Indian Journal of Microbiology, Vol. 48, No. 4, 2008, pp. 453-458. doi:10.1007/s12088-008-0056-8
 M. Hiney, M. T. Dawson, D. M. Heery, R. P. Smith, F. Gannon and R. Powell, “DNA Probe for Aeromonas salmonicida,” Applied and Environmental Microbiology, Vol. 58, No. 3, 1992, pp. 1039-1042.
 M. A. Parker, “Bradyrhizobia from Wild Phaseolus, Desmodium and Macroptilium Species in Northern Mexico,” Applied and Environmental Microbiology, Vol. 68, No. 4, 2002, pp. 2044-2048.
 C. Rachman, P. Kabadjova, R. Valcheva, H. Prévost and X. Dousset, “Identification of Carnobacterium Species by Restriction Fragment Length Polymorphism of the 16S-23S rRNA Gene Intergenic Spacer Region and Species-Specific PCR,” Applied and Environmental Microbiology, Vol. 70, No. 8, 2004, pp. 4468-4477.
 J. Sambrook, E. F. Fritsch and T. Maniatis, “Molecular Cloning: A Laboratory Manual,” 2nd Edition, Cold Spring Harbor Laboratory Press, New York, 1989.
 V. Singh and P. Somvanshi, “Inhibition of Oligomerization of Aerolysin from Aeromonas hydrophila: Homology Modeling and Docking Approach for Exploration of Hemorrhagic Septicemia,” Letters in Drug Design and Discovery, Vol. 6, No. 3, 2009, pp. 215-223.
 V. Singh, P. Somvanshi, G. Rathore, D. Kapoor and B. N. Mishra, “Gene Cloning, Expression and Homology Modeling of Hemolysin Gene from Aeromonas hydrophila,” Protein Expression and Purification, Vol. 65, No. 1, 2009, pp. 1-7. doi:10.1016/j.pep.2008.11.015
 A. J. Martínez-Murcia, L. Soler, M. J. Saavedra, M. R. Chacón, J. Guarro, E. Stackebrandt and M. J. Figueras, “Phenotypic, Genotypic, and Phylogenetic Discrepancies to Differentiate Aeromonas salmonicida from Aeromonas bestiarum,” International Microbiology, Vol. 8, No. 4, 2005, pp. 259-269.
 A. P. Liguori, S. D. Warrington, J. L. Ginther, T. Pearson, J. Bowers, M. B. Glass, M. Mayo, V. Wuthiekanun, D. Engelthaler, S. J. Peacock, B. J. Currie, D. M. Wagner, P. Keim and A. Tuanyok, “Diversity of 16S-23S rDNA Internal Transcribed Spacer (ITS) Reveals Phylogenetic Relationships in Burkholderia pseudomallei and Its Near-Neighbors,” Plos One, Vol. 6, No. 12, 2011, p. e29323.
 M. Laganowska and A. Kaznowski, “Restriction fragment length polymorphism of 16S-23S rDNA intergenic spacer of Aeromonas spp,” Systematic and Applied Microbiology, Vol. 27, No. 5, 2004, pp. 549-557.
 E. Szczuka and A. Kaznowski, “Typing of Clinical and Environmental Aeromonas sp. Strains by random Amplified Polymorphic DNA PCR, Repetitive Extragenic Palindromic PCR, and Enterobacterial Repetitive Intergenic Consensus Sequence PCR,” Journal of Clinical Microbiology, Vol. 42, No. 1, 2004, pp. 220-228.
 V. Singh, D. K. Chaudhary, I. Mani, P. Somvanshi, G. Rathore and N. Sood, “Genotyping of Aeromonas hydrophila by Box Elements,” Microbiology, Vol. 79, No. 3, 2010, pp. 370-373. doi:10.1134/S0026261710030136
 A. Rahmati, M. Gal, G. Northey and J. S. Brazier, “Subtyping of Clostridium difficile Polymerase Chain Reaction (PCR) Ribotype 001 by Repetitive Extragenic Palindromic PCR Genomic Fingerprinting,” Journal of Hospital Infection, Vol. 60, No. 1, 2005, pp. 56-60.
 M. Huynen, R. Gutell and D. Konings, “Assessing the Reliability of RNA Folding Using Statistical Mechanics,” Journal of Molecular Biology, Vol. 267, No. 5, 1997, pp. 1104-1112. doi:10.1006/jmbi.1997.0889
 N. S. Kupriyanova, “Conservation and Variation of Ribosomal DNA in Eukaryotes,” Molecular Biology, Vol. 34, No. 5, 2000, pp. 637-647. doi:10.1007/BF02759600
 D. M. Hillis and M. T. Dixon, “Ribosomal DNA: Molecular Evolution and Phylogenetic Inference,” The Quarterly Review of Biology, Vol. 66, No. 4, 1991, pp. 411-453.
 V. Singh and P. Somvanshi, “Computational Modeling Analyses of RNA Secondary Structures and Phylogenetic Inference of Evolutionary Conserved 5S rRNA in the Prokaryotes,” Journal of Molecular Graphics and Modeling, Vol. 27, No. 7, 2009, pp. 770-776.
 P. Somvanshi, V. Singh and M. Arshad, “Modeling of RNA Secondary Structure of Non Structural Gene and Phylogenetic Analysis of Influenza A Virus through the in Silico Methods,” Journal of Proteomics Bioinformatics, Vol. 1, 2008, pp. 219-226. doi:10.4172/jpb.1000026
 V. J. Pidiyar, K. Jangid, M. S. Patole and Y. S. Shouche, “Analysis of 16S-23S Intergenic Spacer Regions and rrn Operon Copy Number of Aeromonas culicicola MTCC 3249T,” DNA Sequences, Vol. 14, No. 3, 2003, pp. 183-194. doi:10.1080/1042517031000101257