Research Journal of Recent Sciences _________________________________________________ ISSN 2277-2502 Vol. 4(ISC-2014), 14-18 (2015) Res. J. Recent. Sci. International Science Congress Association 14 Identification of Pseudomonas using Probabilistic identification of Bacteria (PIB) Software Bhojiya A.A. and Joshi H.Department of Biotechnology, Mohanlal Sukhadia University, Udaipur-313001, Rajasthan, INDIAAvailable online at: www.isca.in, www.isca.me Received 14th November 2014, revised 25th December 2014, accepted 12th January 2015 AbstractThe genus Pseudomonas is a metabolically versatile group of gram-negative, motile, rod-shaped bacteria. They are common soil-dwelling aerotactic gram-negative proteobacteria with the unique ability to utilize exotic carbon sources for energy. Some members of the genus Pseudomonas are able to metabolize chemical pollutants in the environment, and as a result can be used for bioremediation. Therefore, characterization of various species of Pseudomonas is of significant importance. In the present study four isolates HMR1, HMR4, HMR7 and HMR16 were characterized on the basis of morphological and biochemical characteristics aided with Probabilistic identification of bacteria (PIB) software. For the isolates, HMR1 and HMR16 identification threshold reached to 1.0 and for the isolates, HMR4 and HMR7 identification threshold reached to 0.98906. Isolate HMR1 and HMR16 were identified as Pseudomonas aeruginosa and isolate HMR4 and HMR7 were identified as Pseudomonas putida. Keywords: Pseudomonas, HMR, PIB software, Biochemical characterization, Bioremediation. Introduction The genus Pseudomonas is found ubiquitously in nature with many ecological, economic functions and health-related importance. In the environment, these bacteria are involved in various metabolic activities like cycling of element and the degradation of xenobiotic pollutants. Many Pseudomonas species were recovered from different heavy metal contaminated sites such as soil, sewage, irrigation and agricultural drainage canals1,2. Under laboratory conditions, P. aeruginosa resist high concentrations ofheavy metals like Zn, Cu, Ni, Pb, Cd and Hg3,4,5The taxonomy of the Pseudomonasgenus is complex, consisting of at least 105 recognized species. Many Pseudomonas speciesare metabolically versatile and utilize large number of organic compounds as unique carbon and energy sources. This versatility allows them to survive in many extreme conditions as natural autochthonous microflora making them attractive candidates for use in bioremediation7, 8. An accurate and rapid system for the Pseudomonas identification is essential in order to determine or monitor their role in the environment.Molecular techniques like PCR have been used for microorganism identification; however, they are not without their own limitations i.e. they are too expensive specially species specific PCR and some time it gives false results due to mutations and some other changes. The exquisite sensitivity of PCR is a double edged sword which makes pseudo positive results from even the minutest degree of contamination a serious threat 9, 10. The DNA or RNA extraction method used can also bias diversity studies. Harsh extraction methods like bead beating can shear the nucleic acids which lead to problems in subsequent PCR detection. Various nucleic acid extraction methods will result in different yields of product11. There is a loss of DNA or RNA during subsequent purification steps which again potentially biasing PCR-based diversity studies. Differential amplification of target genes can also bias molecular diversity analysis. Therefore, biochemical characteristics are still the touchstone for bacterial identifications. Biochemical testing aided with probabilistic statistical software12 provides reliable and cheaper method for the identification of bacteria. Probabilistic identification of bacteria (PIBWin) is a windows version of a DOS program PIB (also called Bacterial Identifier), an implementation of Bayes theorem by Willcox et al.13. An identification score was calculated as the Willcox probability P, for identification thresholds of P � 95, P� 98 and P � 99 for all the isolates and reference strains. Lapage14described identification of 1,079 reference and 516 field strains of gram-negative, rod-shaped bacteria using computer-assisted probabilistic method. Joshi and Chaudhary15 used PIB software and identified different species of Lactobacillus with maximum ID score of 0.980. Ottaviani et al. 16 identified Vibrio using the free software probabilistic identification of bacteria with identification thresholds of P � 0.9. Rajput et al. 17 characterized and taxonomically identified various strain of Streptomyces using probabilistic identification of bacteria (PIB) Win software. Carson et al.18 identified motile Aeromonas species with Willcox probability scores of 0.99 using an improved probability matrix. The present study was aimed to identify Pseudomonas bacteria by biochemical tests aided with PIB software. Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 4(ISC-2014), 14-18 (2015) Res. J. Recent. Sci. International Science Congress Association 15 Material and Methods Source and maintenance of bacteria: Heavy metal tolerant bacterial isolates HMR1, HMR4, HMR7 and HMR16 previously isolated from heavy metal contaminated sites of Zawar, Udaipur 19 on nutrient agar medium supplemented with zinc sulphate heptahydrate were used in this study. All the isolates were routinely grown at 37C for 24h on nutrient agar medium supplemented with 1 mM zinc sulphate heptahydrate and stored at -20C in glycerol. Morphological and Biochemical characterization of the isolates: Identification of pure bacterial isolates was performed by studying colony morphology, motility morphological characteristics such as Gram staining and microscopic examination and growth at different temperature. The biochemical tests such as oxidation fermentation reaction (OF) on Hugh and Leifson oxidation-fermentation medium 20, catalase test, oxidase activity, citrate utilization, arginine hydrolysis, starch hydrolysis, lipid hydrolysis, urease activity, malonate utilization, nitrate reduction, gelatin and casein hydrolysis were performed. The growth on MacConkey agar, cetrimide agar and King’s Medium was also checked. PIB software based identification: The data obtained from morphological and biochemical characterization was fed to the identification matrices of known strain. The program provides Identification scores (ID scores) and Identification modal scores (ID modal scores). A threshold of 0.95 (95%) was used as the working confidence level to establish agreement between isolate identification based on conventional methods and that generated by the probability-based method.Results and Discussion Unlike many environmental bacteria, Pseudomonas are of utmost importance and well-studied. These bacteria are of particular concern not only because of their high resistance to heavy metals and other toxic substances, but also for their simple nutritional requirements and rapid growth on standard laboratory media. In the present investigation efforts were made to identify Pseudomonas by series of biochemical test aided with PIB software. Four heavy metal tolerant isolates HMR1, HMR4, HMR7 and HMR16 were characterized on the basis of various morphological and biochemical characteristics (table-1). In Gram’s staining, the morphology of isolated Pseudomonas strains showed Gram-negative, pink colored, medium rod shaped appearance. These findings agreed with the findings reported by earlier researchers21,22. Isolate HMR1 and HMR16 have light brown, flat, small, transparent, round colonies and isolate HMR4 and HMR7 have Off-white, Elevated, Round, small colonies. These characteristics colonies were similar with that of previous studies23,24. All the isolates showed motility at room temperature and 37C. All the isolates gave negative reaction for fermentation of glucose but they gave positive reaction for oxidation of glucose when grown on Hugh and Leifson oxidation-fermentation medium. All the isolates gave positive reaction for catalase activity, oxidase activity, citrate utilization, arginine hydrolysis and negative reaction for amylase activity. Both the isolates HMR1 and HMR16 gave positive result for lipase activity, urease activity, utilized malonate, reduced nitrate and hydrolyzed gelatin and casein whereas isolates HMR4 and HMR7 gave negative results for lipase activity, urease activity, utilization of malonate, nitrate reduction, gelatin and casein hydrolysis. Biochemical characterization of the isolated P. aureginosa and P. putida were similar to previous studies which identified the same organisms from various sources21,25. Growth of these four isolates was further checked on selective and differential media like cetrimide agar and MacConkey agar respectively. All the isolates were able to grow on cetrimide agar. All of them produced colorless colonies on MacConkey agar which indicated that they were lactose non fermenter. In this study both the isolates HMR1 and HMR16 produced green pigment and fluorescence pigment on King’s A medium and King’s B medium respectively. Our results are consistent with Todar26who stated that majority of the Pseudomonas species produce blue-green pigment pyocyanin. The isolates HMR4 and HMR7 fail to produce any pigment when grown on King’s A medium but produced fluorescence pigment on King’s B medium. The results obtained for pigment production are in agreement with the earlier reports27,25. Growth of these four isolates was checked at different temperature like 5C, room temperature, 37 oC and 42 oC. All of them were able to grow at room temperature, 37 oC and 42 oC, but failed to grow at 5C .The above results obtained for morphological and biochemical characteristics were then fed to the identification matrices of known strain in PIB software. The Identification score (ID) and Identification modal score for isolate no. HMR1 and HMR16 were same i.e. 1.0 and 0.29870 respectively (figure 1). The identification score and ID Modal score for isolate no. HMR4 and HMR7 were same i.e. 0.98906 and 0.04167 respectively (figure-1). The analysis revealed that identification score reached the maximum 0.9500 therefore the isolates HMR1 and HMR16 were identified as Pseudomonas aeruginosa and the isolates HMR4 and HMR7 were identified as Pseudomonas putida. These results are parallel to previous studies which identified various species of Pseudomonas using PIB software and obtained identification score in the range of 0.8 to 0.99 28, 29. Our results were in accordance with that of Holmes et al.30 who used computer-based probabilistic method and identified 621 strains of gram-negative, aerobic, non-fermentative bacteria including 20 species of Pseudomonaswith identification score of 0.99. Vinitha Ramanath Pai et al.31identified different species of Pseudomonas using PIB software. They obtained maximum Identification score of 0.80411. In this regard our results are better because high Identification score was obtained for identification of Pseudomonas using PIB software. Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 4(ISC-2014), 14-18 (2015) Res. J. Recent. Sci. International Science Congress Association 16 Table-1 Morphological and biochemical characterization of heavy metal tolerant isolates. Characteristics Isolates HMR1 HMR16 HMR4 HMR7 Motility at 37 o C + + + + Motility at RT + + + + Growth at 5 o C - - - - Growth at RT + + + + Growth at 37 o C + + + + Growth at 42 o C + + + + Gram reaction - - - - Shape rod rod rod rod (O/F) test +/- +/- +/- +/- Catalase + + + + Oxidase + + + + Citrate Utilization + + + + Arginine Hydrolysis + + + + Amylase - - - - Lipase + + - - Urease + + - - Malonate utilization + + - - Nitrate Reduction + + - - Gelatin Hydrolysis + + - - Casein Hydrolysis + + - - Growth on a)MacConkey agar + + + + b)Cetrimide agar + + + + c)King’s A Medium (Pyocyanin) + + No Pigment No Pigment d)King’s B Medium (Fluorescent) + + No Pigment No Pigment + =positive,- =negative, RT=room temperature, O/F= oxidation/fermentation Figure-1 Identification of heavy metal tolerant bacteria by PIB software Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 4(ISC-2014), 14-18 (2015) Res. J. Recent. Sci. International Science Congress Association 17 Conclusion The biochemical testing coupled with a robust identification matrix, provides a convenient basis for identifying unknown bacteria and re-establishes the importance of biochemical tests. Thus this identification system for identifying Pseudomonaswill be of use as these strains could be a potential candidate for heavy metal removal from pollutedsites. Acknowledgement Financial assistance to Ali Asger Bhojiya by the University Grants Commission, India in the form of Maulana Azad National Fellowship (MANF-SRF), and research support from the Department of Biotechnology, Mohanlal Sukhadia University, Udaipur, Rajasthan, India is acknowledged. References1.Shoreit A. and Soltan E., Fluorescent and non-fluorescent Pseudomonas species from Sohag Governorate (Upper Egypt), Bull. Fac. 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