Research Article: Characterization of antibiotic resistant and enzyme producing bacterial strains isolated from the Arabian Sea

Date Published: January 11, 2016

Publisher: Springer Berlin Heidelberg

Author(s): Preeti N. Tallur, Dayanand B. Sajjan, Sikandar I. Mulla, Manjunatha P. Talwar, A. Pragasam, Vinayak M. Nayak, Harichandra Z. Ninnekar, Shivanand S. Bhat.


Marine bacteria are known to produce many bioactive molecules and extracellular enzymes of commercial importance. We have investigated the bacterial diversity of the coastal area of Karwar, Karnataka State, India. Among these bacterial isolates, five bacterial strains were selected and identified by their morphological, biochemical characteristics and phylogenetic analysis based on 16S rRNA gene sequences. The identified bacterial isolates, Bacillus toyonensis PNTB1, Lysinibacillus sphaericus PTB, Vibrio vulnificus PMD, Shewanella MPTDBS, and Pseudomonas chlororaphis PNTB were characterized for their tolerance to salt and antibiotics. Vibrio vulnificus PMD showed maximum tolerance at higher concentration of salt than other bacteria. These bacterial strains were screened for the production of extracellular enzymes such as lipase, cellulase, pectinase, tannase, chitinase, and l-glutaminase. Vibrio vulnificus showed maximum production of l-glutaminase enzyme. Bacillus toyonensis PNTB1 shows lipase, CM-cellulase and chitinase activities. These isolated bacterial cultures were also utilized most of the aromatic compounds at 7 mM. These findings indicate the organisms present in this zone may have more potential applications in bioremediation, agricultural, industrial, and therapeutics.

Partial Text

Marine environment is a source for the isolation of a novel microorganism(s) with the potentiality to produce active secondary metabolites. Among marine organisms, bacteria are of particular interest since they play a vital role in the cycle of matter in water (Rheinheimer 1980). Marine bacteria are well known for their varied bioactive properties, which include the production of secondary metabolites, highly thermostable enzymes and bioactive compounds (Ashadevi et al. 2008). So, it is important to determine the types of bacteria present in the marine ecosystem, the role they play in the functioning of that ecosystem (Nazia and Nuzhat 2006). Hence, in the present study an attempt has been made to isolate and biochemically characterize some of the important marine bacteria from coastal area of Karwar (Karnataka State, India), with a view to identify the organisms and to study their salt tolerance, maximum tolerable concentration of antibiotics, utilization of various aromatic compounds as a growth substrate and also to evaluate their enzyme production potentials. There are reports on the study of marine bacterial diversity from different coastal area of sea (Chiaki et al. 1985; Bozal et al. 2002; Zamudio-Maya et al. 2008; Bal et al. 2009; Aureen et al. 2010; Aisha and Nuzhat 2011). There are very few reports on microorganisms which were isolated from coastal area of Karwar (Pankaj et al. 2011, 2013; Sanjay Kumar and Nagappa 2011; Shreedevi and Rathod 2011). However, there is not much information available on the bacterial cultures isolated from coastal area of Arabian Sea (Karwar, Karnataka State, India) having the ability of producing various extracellular enzymes with maximum tolerance of different antibiotics and salt concentrations and also utilization of various aromatic compounds. Karwar is the coastal city of Arabian Sea and Head Quarters of Uttar Kannada District and is in the West-coast of India, is situated 1315511N latitude and 7.4015–751051E longitude. This place is gained a prominent place in the map of India because of the location of the recently commissioned Indian Naval Base called ‘Sea Bird’ and Kaiga Atomic Power Station in its vicinity. In the present study, we describe the cultural, morphological and biochemical characterization of pure bacterial cultures, isolated from marine sediments, west coastal area of Arabian Sea. Further, they were identified on the basis of 16S rRNA gene sequence analysis. In addition, we also present results of the bacterial cultures having the ability to various extracellular enzyme activities with their maximum tolerance towards various antibiotics and salt concentrations. These pure bacterial cultures were also utilizing various aromatic compounds as a growth substrate.

In the present study five commercially exploitable strains of pure bacterial cultures have been selected for extracellular enzyme activities, utilization of various aromatic compounds as a growth substrate, maximum tolerance towards various antibiotics and salt concentrations studies. All the isolated pure bacterial cultures were gram-positive and gram-negative, rod-shaped and aerobic. The cultural, morphological, and biochemical characteristics of the isolated pure bacterial cultures are given in Table 1. According to Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994) and by phylogenetic analysis based on 16S rRNA gene sequences, the isolates were identified as Bacillus toyonensis strain PNTB1, Lysinibacillus sphaericus strain PTB, Vibrio vulnificus strain PMD, Shewanella strain MPTDBS, and Pseudomonas chlororaphis PNTB. Their sequences were deposited in Genbank under accession number KJ796479, KJ710336, KJ933507, KJ796480, and KJ796478, respectively. The individual pure culture 16S rRNA gene sequence analysis was done at RDP II and NCBI, where relevant sequences from these databases were downloaded for further analysis (Hoskeri et al. 2011). The phylogenetic relationship of isolated individual pure bacterial cultures with other bacterial cultures is shown in Fig. 1. The individual pure bacterial culture is closely aligned with their respective genus between 95 and 100 % (Fig. 1). Also, these pure cultures were analyzed by AFM. AFM is a powerful tool for the analysis of topographical features of bacteria with little or no modification of the sample. It is versatile, robust, fast and economical compared other structural analysis techniques. Vibrio vulnificus PMD was found to be rod shaped with an average length of 442.14 nm and height of 23.178 nm (Fig. 2a). Shewanella strain MPTDBS was found to be rod shaped with an average length of 1.497 µm and height of 130.54 nm (Fig. 2b). Lysinibacillus sphaericus strain PTB has two morphological forms, vegetative rod bacterium, and spherical spores. AFM analysis of spores L. sphaericus have typical spherical shapes with an average diameter of 518.5 nm and height of 61.342 nm (Fig. 2c). Pseudomonas chlororaphis PNTB1 was found to be rod shaped with an average length of 2.498 µm and height of 284.084 nm (Fig. 2d). Bacillus toyonensis strain PNTB1 was found to be rod shaped with an average length of 453.436 nm and height of 9.235 nm (Fig. 2e). Although AFM suffers from minor flaws in the length measurement, due to its probe tip lateral surface interaction with the sample (Kuznetsov and McPherson 2011). Hence, the isolated pure bacterial cultures are the novel strains of their corresponding bacterial genus.Table 1Cultural, morphological and biochemical characteristics of the isolated pure bacteriaCharacteristicsBacillus toyonensis PNTB1Lysinibacillus sphaericus PTBVibrio vulnificus PMDShewanella strain MPTDBSPseudomonas chlororaphis PNTBCultural and morphological characteristics Colony morphologyIrregular, curled, umbonate, rough, turbid white, opaqueRegular, circular smooth, raised transparent, whiteRegular, smooth, circular, concave transparent, pale yellowRegular, circular, transparent glistening, smooth, yellowRegular, entire, opaque, smooth, raised, pale yellow Vegetative cellsRodRodRodRodRod Motility+−+++ Gram reaction++−−− Endospores++−−− Pigment formation−−−−Fluorescent diffusible pigment Temperature (optimum)10–45 °C (37 °C)20–40 °C (30 °C)20–45 °C (30 °C)15–40 °C (35 °C)20–40 °C (30 °C) pH (optimum)5.5–10 (7.1)6–9 (7.2)6–9.5 (7.0)6.5–9.5 (7.2)6–9 (7.0)Biochemical characteristics Catalase+++++ Oxidase+++++ Urease−+++± Lysine decarboxylase++++− Agrinine dihydrolase+–+−− Starch hydrolysis+++−± Gelatin hydrolase++−+− H2S production−−−−− Casein hydrolysis+−−−− Indole production−−−−− Ornithine decarboxylase−−+++ Nitrate Reduction+++++ ONPG−−+−− Citrate utilization+−++− Methyl red test−−−−− Voges Proskauer test+±−+−Oxidation/fermentation (O/F) Mannitol−−O and F−− GlucoseO and FO and FO and F−O and F Lactose−−O and F−−Acid and gas production from carbohydrates Mannitol−/−−/−±−/−−/− Glucose±+/+±−/−± Sucrose±±/−±−/−± Lactose−/−±/−±−/−−/− Fructose±−/−±−/−± Galactose±−/−±±−/− Maltose±−/−±−/−± Xylose−/−−/−±−/−−/− Arabinose−/−±−/−−/−−/−Cultural and morphological characteristics: + present, − absent; Biochemical characteristics: ± present or absent, − no O/F, ± acid production from carbohydrate/no gas production from carbohydrate, −/− no acid/no gas production, +/+ acid/gas production, ±/− acid or no acid production from carbohydrate/no gas production from carbohydrateFig. 2AFM pictures of aVibrio vulnificus PMD, bShewanella strain MPTDBS, cLysinibacillus sphaericus PTB, dPseudomonas chlororaphis PNTB, eBacillus toyonensis PNTB1




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