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International Journal of Environmental Biology

Universal Research Publications. All rights reserved

ISSN 2277–386X
Original Article
Serratia marcescens OU50T sp. nov., a cellulose and pha producing bacterium
isolated from polluted water
Nagamani1 Pammi, Chaitanya2 K, Mahmood1 SK
1
Microbiology Lab, Department of Botany, Osmania University, Hyderabad, 500007, India
2
Research scholar, JNTUH, Hyderabad, India
Corresponding Author: Nagamani P E mail: [email protected] 9492423698
Received 05 June 2015; accepted 22 June 2015
Abstract
Strain Serratia OU50T was isolated from polluted pond water. Cells were characterized by Gram negative, aerobic,
flagellaeted, fluorescent, short rods, capable of accumulate polyhydroxyalkanoates. Grows at 10-400 C, pH 6.0-10.0 and 2-
7% Nacl. Growth occured on trypticase soy agar, nutrient agar, and MacConkey agar. Catalase positive, oxidase negative,
able to degrade chitin and cellulose. Whole cell fatty acids are C10:0 3oH, C12:03OH, C14:0,C14:02OH, C16:0, C17:0cyclo,
C19:0cyclo w 8C,summed feature 2 , summed feature 3 and summed feature 8.G+C content content of the type strain is 55
mol %. Sensitive to neomycin, novobiocin, nalidixic acid, Nitrofurazone and kanamycin, resistant to penicillin, ampicillin,
oxytetracyclin, In the phylogenetic analysis based on the neighbour-joining algorithm, strain OU50T joined the cluster
comprising Serratia marcescens subsp Sakuensis, Serratia nematodiphila, Serratia marcescens subsp. Marcescens,
Serratia ureilytica . Strain OU50T exhibited 16S rRNA gene sequence similarity values of 98.98%, 98.84, 98.64, and
97.81% to the type strains respectively. The 16S rDNA gene sequence was deposited in gene bank with accession number
FN663623. From the above results the isolate was tentatively identified as Serratia marcescens OU50T and was submitted
in the (JCM 17283T =CCM 7838T = CCTCC AB 2010469T)
© 2015 Universal Research Publications. All rights reserved
Keywords:- Serratia marcescens OU50T; Bacterial characterization; PHA: Polluted pond.

1. Introduction in the colonisation of surfaces[4] (Matsuyama, et al.,1992)

Serratia marcescens, a gram-negative, rod shaped bacillus Some members of the genus Serratia also have clinical
classified as a member of the Enterobacteriaceae family, importance[5and6] (Grimont & Grimont, 1992; Brenner,
has been recognised as a cause of hospital-acquired 1984). In this paper, we report the isolation of an aerobic,
infection for the last two decades. It is a widely distributed Gram negative, with flagellum, fluorescent, short rods, red
saprophytic bacterium, and has been found in food, pigmented Catalase, cellulose, lipase positive capable of
particularly in starchy variants which provide, an excellent accumulate polyhydroxyalkanoates (PHA). On the basis of
growth environment. This organism was known formerly phenotypic characteristics, chemotaxonomic and genotypic
by a variety of names, including Chromobacterium data, the isolate is a previously undescribed species of the
prodigiosum [1]. S. marcescens produces several genus Serratia and we propose the name Serratia
extracellular enzymes [2], considered originally to be an marcescens OU50T sp. nov.
innocuous, non-pathogenic saprophytic water organism and 2. Materials and Methods
was often used as a biological marker because of its easily 2.1 Isolation of bacterial strain and culture conditions
recognised red colonies. But S. marcescens has now been Strain OU50T was isolated from polluted pond water from
implicated as an aetiological agent in every conceivable Uppal, located in the city of Hyderabad, India. It was
kind of infection. Aside from, the production of isolated by the standard dilution-plating method on NA
marcescins, S. marcescens is unique among enteric bacteria (nutrient agar) (Himedia). The morphological,
in many respects. It secretes extracellular chitinase, several physiological and biochemical characteristics of strain
proteases, a nuclease and a lipase [3], and produces a OU50T were investigated using routine cultivation on NA
wetting agent or surfactant called 'serrawettin' which helps at 300C. Cell morphology was examined by light

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microscopy and transmission electron microscopy (Hitachi, on 200-mesh copper transmission electron microscopy
H-7500). Cultures of strain OU50T in the lag-, log- and (TEM) grids (Ted Pella) were rendered hydrophilic by
stationary phases of growth were observed under a phase- high-voltage glow discharge. The centrifugation conditions
contrast microscope (1000×) to find out their shape and used ranged from 10,000 to 41,000 rpm for 0.5 to 4 h in a
motility. Presence of PHA granules were confirmed by swinging bucket rotor (type SW41; Beckman). The bacteria
florescence microscopy. Biochemical characteristics were on grids were stained by submerging the grids for 20 s in
checked with the Hi25 Enterobacteriaceae identification 0.5% (wt/vol) uranyl acetate and then were rinsed three
kit(KB003) and Hi Carbohydrate kit parts A, B and C times (10 s each) by submersion in aliquots of Milli-Q
(KB009) (both from Hi- Media) according to the water. The grids were examined with a Hitachi model H-
manufacturer’s protocol. Other tests were performed by 500 TEM by using an accelerating voltage of 80 to 100 kV.
growing the culture at 300 C in the appropriate medium. 2.5 Sequencing of 16S rDNA
The activities of catalase, oxidase, phosphatase, gelatinase, Chromosomal DNA was isolated and purified according to
urease, cellulase, protease, lipase, lecithinase, were the method described by Yoon et al., [13]. Amplification of
determined according to standard methods [7]. Utilization the 16S rRNA gene of strain OU A7T by PCR (primers 27F
of different sugars, leading to the formation of acid with or and 1492R) was carried out as described by Lane,
without gas production was monitored [8]. Gram staining 1991[14]. The amplified product was directly sequenced on
was performed by the Burke method [9]. Motility was a 3100 automatic DNA sequencer (Applied Biosystems).
assessed on 0.4% nutrient agar plates and also by hanging Pairwise sequence similarities were calculated by using a
drop method [10]. Spore staining was done using Schaeffer global alignment algorithm, which was implemented using
& Fulton’s spore-staining kit (K006-1KT; HiMedia) the EzTaxon server. Phylogenetic analyses were performed
according to the manufacturer’s protocol. Sensitivity of using the software package MEGA 4.0 [15] after multiple
cultures to different antibiotics were determined by alignments of the sequence data with CLUSTAL_X [16].
previously described methods[11].Biochemical Distances were calculated using distance options according
characteristics were also checked with the Hi25 to Jukes & Cantor, 1969[17] and clustering was performed
Enterobacteriaceae identification kit (KB003) and Hi using the neighbour joining method [18]. Bootstrap
Carbohydrate kit parts A, B and C (KB009) (both from analyses were used to evaluate the tree topology by means
HiMedia) according to the manufacturer’s protocol. of 1,000 resamplings[19].
Sensitivity of the culture to nine antibiotics was determined 2.6 Cellular fatty acid analysis
using antibiotic discs(Hi Media), containing: polymyxin For cellular fatty acid analysis, cell mass of strain OU50
B(100 IU ml-1), penicillin G(10 IU ml-1), ampicillin (10 µg was harvested from NA plates after incubation for 24 hours
ml-1), novobiocin (5 µg ml-1), tetracycline(50 µg ml-1), at 300C. The fatty acids were extracted and fatty acid
kanamycin(30 µg ml-1), neomycin(50 µg ml-1), methyl esters were prepared according to the standard
nitrofurazone(30 µg ml-1),and nalidixicacid(50 µg ml-1). protocol of the MIDI/Hewlett Packard Microbial
2.2 Scanning electron microscopy Identification System [20]. The resulting profles were
Samples were fixed in 2.5% Gluteraldehyde in 0.1 M identified with the Microbial Identifcation software (MIDI)
phosphate buffer (pH 7.2) for 24 hrs at 4oC and post fixed using the RTSBA database (version 6.0) (Microbial ID,
2% in aqueous osmium Tetroxide for 4 hrs, in the same Newark, DE, USA).
buffer. After the post fixation samples were dehydrated in 2.7 Determination of of DNA G+C content
series of graded alcohols and dried to critical point drying The DNA G+C content was determined by the method of
with electron microscopy Science CPD unit. The dried [21] with the modification that DNA was hydrolysed and
samples were mounted over the stubs with double-sided the resultant nucleotides were analysed by reversed-phase
carbon tape. Applied a thin layer of gold coat over the HPLC.
samples by using an automated sputter coater (JEOL JFC- 3. Results and discussion
1600) for 3 min. Scanned the samples under Scanning The results of cultural, morphological and biochemical
Electron Microscope (Model, JOEL_JSM 5600) at various characteristics of OU50T were used to tentatively classify
magnifications. the isolate using Bergey’s Manual of Systemic
2.3 Polymer extraction Bacteriology [22]. Photographs of biochemical tests, and
The polymer was extracted from the bacterial pellet by extra cellular enzymes were shown in the Figure 1. (a-h).
using the hypochlorite method [12]. Polymer was washed Figure 2 include sensitivity and resistance of OU50T
with methanol and acetone consecutively and centrifuged at towards various antibiotics. OU50T was resistant to
8000 rpm for 20 min, was dissolved in hot chloroform Penicillin, Ampicillin, Oxytetracycline, Neomycin and
(60° C) and the solution poured onto glass trays. The sensitive to Nalidixic acid, Nitrofurazone and Kanamycin.
chloroform was allowed to evaporate slowly at 4° C by Figure 3. is showing the morphology of the isolates under
placing the trays in the cold room, because uniform films of scanning electron microscope. Figure 4 is showing the
the polymer could not be obtained at room temperature, due presence of flagella under transmission electron
to rapid evaporation of chloroform. On evaporation of microscope. Figure 5 and 6 are showing biochemical
chloroform, the film of the polymer was obtained, properties and Carbohydrate utilization testsof the isolate.
2.4 Transmission Electron Microscopy to detect flagella PHA granules were present with in the cells (Fig. 7)
Bacteria were preserved by adding formaldehyde (final Though, the recent trend of confirming the placement of
concentration, 2%). Carbon- stabilized Form var supports cultures in taxonomic groups is based only on the

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 Figure 5: Biochemical tests
1. ONPG β galactosidase2. Lysine decarboxylase3. Ornithine
decarboxylase 4. Urease5. Phenylalanine deamination 6. Nitrate
reduction 7. H2S production 8. Citrate utilization 9.Voges
Proskauer’s 10. Methyl red 11. Indole 12. Malonate.

Figure 6: Carbohydrate utilization tests for OU50T;

Positive-Yellow; Negative-Pink
1. Esculine 2.Arabinose 3.Xylose 4. Adonitol 5.Rhamnose
6.Cellobiose 7.Melibiose 8.Saccharose 9.Raffinose 10.Trehalose
11.Glucose 12.Lactose
Figure 1: Growth and enzyme activities of strain OU50T

Figure 2: Antibiotic sensitivity tests for OU50T

Key:1. Neomycin 2. Penicillin 3. Novobiocin 4. Ampicillin 5.
Nalidixic acid 6. Nitrofurazone 7. Kanamycin 8. Oxytetracyclin

Figure 7: Flourescence microscopy of OU50T showing

strong orange flourescence against dark green back ground
molecular tools, the strain was identified on the basis of
the cultural, morphological and biochemical characteristics
as per the second edition of Bergey’s manual of systemic
bacteriology [22]. Almost complete 16S rRNA gene
sequence of strain OU50T determined in this study
comprised 1484 nucleotides. The phylogenetic analyses
based on 16S rRNA gene sequences revealed that strain
Figure 3: SEM of OU50T OU50T fell within the clade comprising species of the
genus Serratia (Fig 8). In the phylogenetic analysis based
on the neighbour-joining algorithm, strain OU50T joined
the cluster comprising Serratia marcescens subsp
Sakuensis KREDT (AB061685) Serratia nematodiphila
DZ0503SBS1T(EU036987), Serratia marcescens subsp.
Marcescens DSM 30121T(AJ233431), Serratia ureilytica
NiVa 51T(AJ854062) (Fig. 8). Strain OU50T exhibited
16S rRNA gene sequence similarity values of 98.98%,
98.84, 98.64, and 97.81% to the type strains respectively.
3.1 Species description
Strain Serratia OU50T was isolated from polluted water.
Cells were characterized by Gram negative, aerobic, with
flagellae, fluorescent, short rods, capable of accumulate
Figure 4: TEM of OU50T PHA. Red pigmented, circular colonies on nutrient agar

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Figure 8: Phylogenetic position of the bacterium OU50T with the genus Serratia with EMBL/GENE bank Accession No
FN663623
Acknowledgements
smooth with entire margins. Grows at 10-400c, pH 6.0-10.0 The author (P.Nagamani) thanks the University of Grant
and 2-7% Nacl. Growth occured on trypticase soy agar, Commission (UGC), Govt. of India for providing a
nutrient agar, and MacConkey agar. Catalase positive and fellowship.
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Source of support: Nil; Conflict of interest: None declared

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