Chapter 5 Diversity of Culturable Gram Negative Bacteria...

Chapter 5

Diversity of Culturable Gram Negative Bacteria in water at

Kumarakom region of Vembanadu lake

Chapter 5 Diversity of Culturable Gram Negative ……….. of Vembanadu Lake

Characterization and Inventorization of Wetland Microflora along Vembanadu Lake

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5.1 Introduction

Water bodies are natural environment for various groups of organisms, including

microorganisms, to inhabit and develop. Bacteria occur in all types of surface waters and live

in every zone of water bodies, from the surface layer to the bottom deposits. They inhabit

clean and heavily polluted waters, fresh inland waters and the salt water of seas, stagnant

water and watercourses. Microorganisms inhabiting the above water bodies belong to

various groups, genera and species. They frequently come from different environments. The

different conditions prevailing in water mean that the bacteria inhabiting it differ from those

occurring in soil, air or in the digestive tract of organisms. The water itself affects the

organisms existing in it through its physical and chemical properties. Apart from autotrophic

forms, one can find heterotrophic and parasitic forms in water bodies, including those that

are pathogenic for humans and animals (Paluch, 1973). Microorganisms in water bodies

carry out specific biochemical processes, forming groups with specific physiological

properties.

Typically aquatic heterotrophic microflora includes numerous motile organisms,

monotrichately or lophotrichately flagellated, numerous spirochetes (Spirochaeta), Spirillum

and Vibrio as well as the majority of Gram-negative rods of the Pseudomonas,

Achromobacter and Flavobacterium genera (Paluch, 1973; Kunicki-Goldfinger, 1994).

Gram-negative bacteria, do not retain crystal violet dye in the Gram staining protocol.

Volume 1 of Bergey's Manual describes 10 groups of Gram-negative bacteria having

general, medical, or industrial importance. There is considerable morphological and

physiological diversity among the heterotrophic Gram negative Eubacteria. Most of the

Gram-negative, chemoheterotrophic bacteria are grouped under the phylogenetic group

Proteobacteria (the largest taxonomic group). The remaining ones as other heterotrophic

Eubacterial groups.

The Proteobacteria (purple bacteria) group includes: alpha subdivision (which

includes Agrobacterium sp., Brucella sp., the purple non-sulfur bacteria, and the rickettsias),

beta subdivision (which includes Bordetella sp., Neisseria sp., and some Pseudomonas sp.),

gamma subdivision (which includes Family Enterobacteriaceae, Haemophilus sp.,

Legionella sp., Pasteurella sp., Vibrio sp., the purple sulfur bacteria, and some Pseudomonas



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sp.), delta subdivision (which includes the Bdellovibrio, Desulfovibrionales and

Myxococcales) and epsilon subdivision (which include Helicobacter sp.).

Facultatively anaerobic Gram-negative rods are medically relevant group of bacteria.

There are three families making up the bulk of facultative anaerobic Gram-negative rods (39

of 46 genera): these are the family Enterobacteriaceae, family Vibrionaceae and family

Pasteurellaceae.

Family Enterobacteriaceae includes various genera such as Citrobacter,

Enterobacter, Erwinia, Escherichia, Klebsiella, Proteus, Providencia, Salmonella, Serratia,

Shigella and Yersinia, while family Vibrionaceae includes genera such as Aeromonas,

Enhydrobacter, Photobacterium, Plesiomonas and Vibrio. Family Pasteurellaceae includes

three genera such as Actinobacillus, Haemophilus and Pasteurella.

Obtaining data on biodiversity and distribution of various bacterial groups in water

bodies is an essential part of ecological monitoring. Almost 90-95% Gram negative bacteria

are considered to be harmful for the host. Fresh water is host to numerous microorganisms

that affect human health directly. The present study was therefore targeted to determine the

diversity of culturable Gram negative bacteria in the water at Kumarakom region of

Vembanadu lake.

5.2 Review of Literature

In sanitary-bacteriological practice, it is a generally accepted procedure to define

water quality in open water bodies with regard to its bacterial contamination by the

availability of sanitary-indicative microorganisms. At present, mainly quantitative indices

are used that do not reflect variations in microbiocenoses (Nemtseva and Misetov, 2000),

whereas bacterial communities are the most sensitive indicators of any changes in the

environment.

O’Sullivan et al. (2002) investigated about the bacterial diversity of River Taff

epilithon in South Wales. 16S ribosomal DNA (rDNA) clone library was constructed and

analyzed by partial sequencing of 76 of 347 clones and hybridization with taxon-specific

probes. The epilithon was found to be very diverse, with an estimated 59.6% of the bacterial

populations not accounted for by these clones. Members of the Cytophaga-Flexibacter-



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Bacteroides division (CFBs) were most abundant in the library, representing 25% of clones,

followed by members of α-Proteobacteria, γ-Proteobacteria, Gram-positive bacteria,

Cyanobacteria, β-Proteobacteria, δ-Proteobacteria, and the Prosthecobacter group.

Drucker and Panasyuk (2006) researched on the biodiversity and distribution of

Enterobacteriacae and a nonfermenting group of bacteria in Lake Baikal that belong to

potentially pathogenic bacteria. They correlated the abundance of these pathogenic bacterial

groups with anthropogenic load.

Later Rabeh and Fareed (2008) enumerated and identified most common pathogenic

Gram negative bacteria from the lake Qarun. They identified the presence of Escherichia

coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus vulgaris etc. based on their

physiological and biochemical characters. The bacterial diversity of lake Martel (Mallorca

Island, Spain), the longest known subterranean lake, has been studied by Rivas et al. (2009).

The results obtained showed the complexity of bacterial populations living in Martel

ecosystem because the strains isolated belong to very divergent phylogenetic groups mostly

within Proteobacteria and are related to bacterial species found in water related sources.

Most of the studies of Gram negative bacteria related to the ecological (the presence

and quantity of more important physiological groups of bacteria which indicate the

specificity of the organic pollutants) and sanitary conditions of the water (coliform bacteria,

fecal indicators). These studies are explained below:

5.2.1 Characterization and Prevalence of Enterobacteriaceae

The family of bacteria referred to as the Enterobacteriaceae contains some of the best

known and most thoroughly studied organisms. They are distributed world-wide and exhibit

substantial diversity in ecology, host range and pathogenic potential for man, animals,

insects and plants. The predominant aerobic bacterial flora of the large intestines of human

beings and animals composed of nonsporing, nonacid fast, Gram negative bacilli. They

exhibit general morphological and biochemical similarities and are grouped together in the

large and complex family Enterobacteriaceae. Members of this family may or may not be

capsulated and are motile by peritrichate flagella, or are nonmotile. They are aerobic and

facultatively anaerobic, grow readily in ordinary media, ferment glucose producing acid and

gas or acid only, reduce nitrates to nitrites, and form catalase but not oxidase. Within the



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family, they exhibit very wide biochemical and antigenic heterogenicity. Though the family

is subdivided into groups or tribes, genera, subgenera, species and types, many strains are

met with that possess every conceivable combination of characters and do not fall into any

such arbitary taxonomic category. Bergey’s Manual divides the Enterobacteriaceae family

into five tribes. Escherichieae (containing the genera Escherichia, Aerobacter, Klebsiella,

Paracolobactrum and Alginobacter); Erwineae (Erwinia); Serrateae (Serratia); Proteae

(Proteus) and Salmonelleae (Salmonella and Shigella).

The economic importance of many members of the family together with their ability

to grow on simple laboratory media have made them the object of intense laboratory study

by practitioners of different disciplines including medicine, veterinary science,

epidemiology, agriculture, food and water microbiology, biochemistry and genetics. These

studies have generated a large amount of information on the Enterobacteriaceae but they

have also led to a great deal of confusion from the standpoint of classification and

nomenclature. The family Enterobacteriaceae as currently described comprises Gram-

negative, non-sporing, rod-shaped bacteria that are often motile, usually by peritrichously

arranged flagella. Capsulated and non-capsulated forms occur. Currently there are 28 named

genera in the Enterobacteriaceae but it can be confidently predicted that this number will

increase in the near future.

Enterobacteriaceae are large Gram negative rods usually associated with intestinal

infections. But can be found almost all natural habitats. They are the causative agents of such

diseases as meningitis, dysentery, typhoid and food poisoning.

Klebsiella sp. are widely recognized as opportunistic, antibiotic-resistant pathogens

often acting as agents of respiratory and genito-urinary infections, particularly in patients

under stress. Reports indicate that Klebsiella sp. have been isolated from lakes (Campbell et

al., 1976), rivers (Seidler, 1975), seawater (Dufour and Cabelli, 1974), sewage and various

waste waters (Dufour and Cabelli, 1976; Knittel et al., 1977), and drinking water (Bagley

and Seidler, 1978). Bagley and Seidler (1978) have developed a selective medium (Mac

Conkey-inositol-carbenicillin-agar medium) for Klebsiella enumeration. Niemela and

Vaatanen (1982) investigated survival of Klebsiella pneumoniae in freshwater lake, at

various distances from point of effluent discharge from a paper mill.



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The bacterium Escherichia coli, a very common waterborne commensal/pathogen.

Humans have a large and harmless population of E. coli in their lower, large intestines and

bacteria make up a large fraction of the volume of human feces. When released into drinking

water or recreational water sources, E. coli can be ingested and enter the upper small

intestine, causing diarrhea. Hatha et al. (2004) reported the prevalence of pathogenic

serotypes of E. coli from coastal areas. Their study revealed the presence of more than 40

serotypes of E. coli in the Cochin estuary. Also the prevalence of E. coli, Vibrio cholerae,

Vibrio parahaemolyticus and Salmonella were analysed in the Kumarakom region of

Vembanadu lake (Hatha et al., 2008). Their study revealed substantially high level of

indicator bacteria in the Vembanadu lake during monsoon months.

Brzezinska and Donderski (2006) reported the dominance of Enterobacter aerogenes

(28%), Aeromonas sp. (25%) and Chromobacterium sp. (16%) in oligo–mesotrophic lake.

The quantity and quality composition of the family Enterobacteriaceae bacteria, their spatial

distribution (particularly focusing on the sites where black cormorants tend to dwell) and

seasonal variations in two consecutive years (2000 and 2001) were assayed in the water of

Dlugie Wigierskie Lake, near to Wigry National Park (Wisniewska et al., 2007). Pontes et

al. (2007) characterized a population of Enterobacter sp. of the Enterobacter cloacae

complex isolated from an oligotrophic lake; most isolates were identified by them as

E. cloacae. Fingerprinting polymerase chain reaction (PCR), along with morphological,

biochemical, physiological, and plasmid profiles analyses, including antimicrobial

susceptibility testing, were performed by these researchers on 22 environmental isolates.

Gotkowska-Plachta (2008) reported the presence of Serratia marcescens, Serratia

rubidaea and E. aerogenes in the water samples of Lake Hancza – the deepest lake in

Poland. E. cloacae are facultative anaerobic Gram-negative bacillus belonging to the family

of Enterobacteriaceae. The nomenclature (taxonomy) of the E. cloacae complex is mainly

based on whole genome DNA-DNA hybridizations and phenotypic characteristics (Brenner

et al., 1982; Mehlen et al., 2004). Currently, 6 species have been assigned to the

Enterobacter cloacae complex, including Enterobacter asburiae, Enterobacter cloacae,

Enterobacter hormaechei, Enterobacter kobei, Enterobacter ludwigii and Enterobacter

nimipressuralis. Isolates of the Enterobacter cloacae complex have been increasingly

isolated as nosocomial pathogens, but phenotypic identification of the E. cloacae complex is

unreliable and irreproducible (Paauw et al., 2008).



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Onyuka et al. (2011) determined the prevalence and antimicrobial susceptibility

patterns of Escherichia coli, Salmonella enterica serovar Typhimurium and Vibrio cholerae

O1 isolated from water and two fish species Rastrineobola argentea and Oreochromis

niloticus collected from fish landing beaches and markets in the Lake Victoria Basin of

western Kenya. Studies of Abhirosh et al. (2011) revealed the presence of high density of

faecal coliform bacteria and prevalence of multi drug resistant E. coli and Salmonella

serotypes in the Vembanadu lake, and they concluded that, they may pose severe public

health risk through related water borne and food borne outbreaks.

5.2.2 Characterization and prevalence of Alcaligenes

The strains of Alcaligenes latus were identified as nitrogen-fixing bacteria by Malik

et al. (1981). Alkaligenes sp. is reported as a predominant microbial flora of Escravos river,

found mostly within the discharge zone of produce water effluents (Okoro, 1999). Ansede et

al. (2001) assessed the phenotypic and phylogenetic diversity of dimethyl sulfide-producing

bacteria from salt marsh and adjacent estuarine waters. Phylogenetic analysis of 16S rRNA

gene sequences showed that all of the isolates were in the group Proteobacteria, with most

of them belonging to α and γ subclasses. Only one isolate was identified as a β-

proteobacterium, and it had >98% 16S rRNA sequence homology with a terrestrial species

of Alcaligenes faecalis. Most Alcaligenes strains have been isolated from wastewater

treatment plants or bioremediation sites of contaminated soils because of their degradative

capabilities, which suggests that further study of this genus may help to understand the

ecological diversity of these organisms and their usefulness as biodegradation bacteria

(Fang-Bo et al., 2008). Salehizadeh and Mohammadizad (2008) described that Alcaligenes

faecalis was able to produce biosurfactant after growing on molasses. Okoro and Amund

(2010) isolated Alkaligenes sp. from Escravos River receiving produce water discharge and

studied about the degradation of total petroleum hydrocarbons in produce water by the

isolated Alkaligenes sp.

5.2.3 Characterization and prevalence of Aeromonas

Members of the genus Aeromonas, belonging to the class Gammaproteobacteria, are

Gram-negative, non-spore-forming bacilli or coccobacilli and are facultatively anaerobic,

chemo-organotrophic, oxidase- and catalase-positive, resistant to the vibriostatic agent



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O/129 (2,4-diamino-6,7- diisopropylpteridine), generally motile by means of a single polar

flagellum and are able to reduce nitrate to nitrite (Minana-Galbis et al., 2007). Aeromonads

are primarily aquatic, widespread in environmental habitats, frequently isolated from foods

and often associated with aquatic animals and some species are primary or opportunistic

pathogens in invertebrates and vertebrates including humans (Martin-Carnahan and Joseph,

2005). Aeromonas hydrophila was first proposed as a potential cause of enteric diseases as

early as 1937. Since 1980’s clinical studies had supported the suspicion that at least some

strains of Aeromonas sp. are intestinal pathogens. The species A. hydrophila, A. caviae and

A. sorbia are motile and have been linked to two major groups of human diseases:

septicemia and gastroenterititis (Merino et al., 1995).

Aeromonas sp. are widely distributed in the aquatic environment (Parveen et al.,

1995), seawater (Hazen et al., 1978; Kaper et al., 1981; Martinez-Manzana et al., 1984) and

freshwater ecosystems to which they are indigenous and where they can multiply under

appropriate conditions (adequate temperature, nutrients availability, etc.) (Rippey and

Cabelli, 1979). Cavari et al. (1981) reported that when Aeromonas sp. enter bodies of water

in temperate environments, via sewage effluent or other sources, a significant decrease in the

number of viable cells can occur during those seasons of the year when the water

temperature is low. Parveen et al. (1995) isolated and studied seasonal distribution of

Aeromonas sp. from samples such as water, sediment, plant and plankton samples in the

Dhanmondi lake and the Buriganga river of Bangladesh, throughout the year. The seasonal

distribution of Aeromonas sp. particulariy A. hydrophila and their relationship with physico-

chemical properties of water were studied by several investigators (Burke et al., 1984; Islam

et al., 1992). Previous studies have demonstrated that the most common Aeromonas sp. in

warm polluted waters in the eastern United States is A. hydrophila (Joseph et al., 1979;

Seidler et al., 1980), whereas the majority of Aeromonas from human infections is A. sobria

(Daily et al., 1981).

Bernagozzi et al. (1995) determined the quantity of Aeromonas sp. and Aeromonas

hydrophila in samples of fresh surface water, brakish and seawater with different degrees of

pollution. Marcel et al. (2002) isolated and characterized Aeromonas species in the Ebrie

lagoon over a one year period. Aeromonas species are frequently isolated by them from the

Ebrie lagoon urban area during the rainy and flood seasons when the salinity is low and



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below 10‰. They also showed lower isolation frequency during the dry season when water

salinity is over 10‰.

Brzezinska and Donderski (2006) reported the dominance of A. hydrophila (46%)

and Aeromonas sp. (15%) in eutrophic lake. Being the autochthonous flora of natural waters,

Aeromonads are also found in drinking water supplies worldwide. Egorov et al. (2011)

studied about the occurrence of Aeromonas sp. in drinking water distribution systems in

United States. The results of their survey demonstrated the importance of maintaining

adequate residual chlorine and low turbidity for preventing drinking water contamination

with Aeromonads.

5.2.4 Identification and prevalence of Vibrio species in aquatic ecosystem

The genus Vibrio, within the family Vibrionaceae, is a diverse group of Gram-

negative bacteria found exclusively in the aquatic environment. Important pathogenic

members include Vibrio cholerae, the causative agent of cholera, and Vibrio

parahaemolyticus and Vibrio vulnificus, which have been implicated in diarrhea, septicemia

and wound infections (Farmer et al., 2003). Vibrio cholerae has been isolated with

increasing frequency from continental water, as well as from estuarine water and seawater in

many different geographic areas (Colwell et al., 1977; Muller, 1977; Kaper et al., 1979;

Bashford et al., 1979; Colwell et al., 1981; Lee et al., 1982; West and Lee, 1982; Thomson

et al., 1998). There are descriptions of Vibrio O1 isolates from natural waters (Rogers et al.,

1980). The ability of such environmental strains to produce enterotoxin has been extensively

demonstrated by different assays (Ohashi et al., 1972; Zinnaka and Carpenter, 1972).

The bacterium Vibrio cholerae, while rare in the United States, remains a significant

source of disease and death in countries without proper sewage treatment and potable water

supplies. For example, a cholera epidemic in 1991 killed more than a thousand people in

Peru (South America), where more than 150,000 cases of the illness were confirmed (Clark

and Robinson, 2011). Gray et al. (1985) reported most probable numbers of Vibrio cholerae

and related vibrios in Albufera Lake, Valencia, Spain, and in coastal waters under the

influence of the lake discharges over the course of an annual cycle. They also reported that

the recovery of vibrios was significantly influenced by temperature of water and the type of

water analyzed. Tamplin et al. (1991) described an enzyme immunoassay (EIA) and culture

techniques for the identification of V. vulnificus in seawater, sediment and oysters.



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V. vulnificus, a halophilic marine Vibrio, is an opportunistic human pathogen that can

cause severe wound infections and septicemias with mortalities as high as 60 % (Oliver,

1989; Hlady and Klontz, 1996). Radu et al. (2000) isolated V. vulnificus from coastal water

and were characterized for their antimicrobial resistance, plasmid profiles and were typed by

the PCR-based techniques: a random amplification of polymorphic DNA (RAPD) method

and the enterobacterial repetitive intergenic consensus sequence (ERIC) method.

Amirmozafari et al. (2005) investigated about the occurrence of potentially pathogenic

species of Vibrio in sea water and estuarine environments of the Caspian Sea in the Golestan

province of Iran.

Tudor et al. (2007) investigated the frequency of bacterial species of noncholeric

Vibrio from aquatic samples. They identified the presence of V. alginolyticus, V.

parahaemolyticus and V. vulnificus in the aquatic samples. Chandran et al. (2008) reported

about the prevalence of faecal indicator bacteria, E. coli and pathogenic bacteria, V.

cholerae, V. parahaemolyticus and Salmonella in Vembanadu lake.

The classic strain of V. cholerae agglutinates with O1 antiserum and is usually found

in salty or brackish estuarial waters. Infection is associated with ingestion of contaminated

foods with subsequent severe diarrheal illness. V. cholerae O1 is responsible for the life

threatening secretory diarrhea, mostly associated with epidemic outbreaks when sanitary

conditions are not optimum (World Health Organization, 2010), and the Asiatic cholera

outbreaks have been linked to consumption of unsafe food and water such as drinking lake

and river water, food sold by the roadside and feasting at funeral gathering (Shapiro et al.,

1999; Acosta et al., 2001).

Whitehouse et al. (2010) developed an assay that can identify all known pathogenic

Vibrio species and field-tested it using natural water samples from both freshwater lakes and

the Georgian coastal zone of the Black Sea. This study marks the first application of this

technology to Vibrio sp. and the first use of the Ibis T5000 platform for the direct detection

of bacterial pathogens from natural aquatic environments. Walker et al. (2010) reported a

case of non-O1 V. cholerae urinary tract infection associated with freshwater exposure. The

potential for vibrios to grow in brakish water was also noted by them.



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V. fluvialis is an important cause of cholera-like bloody diarrhea and causes wound

infection with primary septicemia in immunocomprised individuals from developed to

underdeveloped countries, especially in regions with poor sanitation. Igbinosa and Okoh

(2010) documented high densities of disease causing V. fluvialis in the watersheds. Nigro et

al. (2011) investigated the abundance, distribution and virulence gene content of V. cholerae,

V. parahaemolyticus and V. vulnificus in the waters of southern Lake Pontchartrain in

Louisiana, USA, on four occasions from October 2005 to September 2006, using selective

cultivation and molecular assays. They found that the percentage of the B-type V. vulnificus

was significantly higher in the lake in October 2005 (35.8% of the total) compared to other

sampling times (p0.004), consistent with the view that these strains represent distinct

ecotypes.

5.2.5 Characterization of Pseudomonas species

Pseudomonads are opportunistic Gram negative pathogens, naturally occurring in

aquatic environment and as a part of normal gut flora of healthy fish, it cause outbreak when

the normal environmental conditions changed (Angelini and Seigneur, 1988; Roberts, 1989).

Pseudomonas fluorescens, Pseudomonas angulliseptica, Pseudomonas aeruginosa and

Pseudomonas putida were identified in various species of fish as causative agents of

Pseudomonas septicemia (Post, 1987).

Historically, the genus Pseudomonas has been poorly investigated in terms of the

phylogeny of its species. In recent years, some bacterial species that previously had unclear

taxonomic positions within this genus have been reclassified using a polyphasic taxonomic

approach (Anzai et al., 2000). Eissa et al. (2010) isolated Pseudomonas sp. from liver,

spleen, kidneys and gills of Oreochromis niloticus from Qaroun and El Rayan lakes on

Pseudomonas agar media. The cultures were identified based on their morphological and

biochemical characters as Pseudomonas fluorescens biovar I, II, III, Pseudomonas

anguilliseptica, Pseudomonas putida and Pseudomonas aeruginosa.

5.3 Objectives

The microorganisms in the water environment have chief role in the process of

transformation of organic material and in the functioning of the ecosystem as a whole.

Knowing the composition and the dynamics of their population is a realistic indicator for



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determination and forecast of the condition in the aquatic ecosystem. The bacteria are very

important indicators for determination of the level of cleanliness of the analyzed water. As

first pointers for euthrophication, they have primary importance within the frames of the

hydrobiological research. The aim of the present study was to determine the diversity of

culturable Gram-negative heterotrophic bacteria occurring in the water at Kumarakom region

of Vembanadu lake and to identify some of their physiological properties. Specific

objectives are as follows:

1. To identify various culturable heterotrophic Gram-negative bacterial genera isolated

from water of Kumarakom lake up to their species rank, based on culture dependent

phenotypic characteristics.

2. To study the biochemical and physiological properties of the Gram–negative genera

isolated from the water samples of Kumarakom lake.

3. To find out the spatio-temporal variation in the distribution of identified species of

Gram-negative bacteria at selected sites of Kumarakom lake.

5.4 Materials and Methods

Gram-negative bacterial strains isolated from water samples of Kumarakom lake (the

detailed procedure for the sample collection, isolation and generic identification of bacterial

isolates is given in the Chapter 3, sec. 3.4.1., 3.4.3. and 3.4.4.) were grouped into different

generic groups (such as Enterobacteriaceae, Alcaligenes, Aeromonas, Pseudomonas and

Vibrio) based on their Gram reaction, motility, presence of oxidase enzyme and oxidative-

fermentative ability. They were further characterized based on selected tests for each group

as per Bergey’s Manual of Determinative Bacteriology, 9th edn (Holt et al., 2000).

5.4.1 Biochemical differentiation of the species of the family Enterobacteriaceae

Members of the family Enterobacteriaceae were identified as distinct species as per

their biochemical and physiological properties. The following tests were carried out to

determine the species level identity.

5.4.1.1 Methyl red test: In this test the bacteria’s ability to produce mixed organic acids

(lactic, acetic and formic) and the subsequent reduction of pH below 4.6 resulting from acid

fermentation of dextrose was checked. Pure cultures were inoculated into MR-VP broth and



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incubated at 37oC for 24-48 hrs. After incubation a few drops of methyl red reagent was

added. A persistent red colour on addition of methyl red indicated positive for methyl red

test. Yellow or orange colour is considered as negative for methyl red test.

5.4.1.2 Hydrogen sulfide production: The ability of the cultures to produce hydrogen sulfide

was tested by using Triple Sugar Iron (TSI) Agar. In this test the ability of the organism to

ferment three different sugars such as lactose, sucrose and dextrose were analyzed. Cultures

were inoculated into TSI agar by stabbing the butt and streaking the slant and incubated at

37oC for 24 hrs. The production of alkaline (red) slant and acid (yellow) butt indicate the

glucose fermenting ability of the organism, while acid (yellow) slant and butt indicates

utilization of two (lactose and/or sucrose) sugars. The H2S production is indicated by the

blackening of the medium.

5.4.1.3 Lysine decarboxylase: Lysine decarboxylase broth inoculated with pure culture were

incubated at 37oC for 24-48 hrs. If L-Lysine is decarboxylated to cadaverine, there will be an

alkaline reaction and indicator colour then change to purple.

5.4.1.4 Arginine dihydrolase: Arginine dihydrolase semisolid agar is used for detection of

arginine dihydrolase producing microorganisms. Cultures were aseptically stabbed in to the

medium and are incubated at 37oC for 24-48 hrs. Bacteria producing arginine dihydrolase

enzyme in this medium produces alkaline products and elevates the pH. Bromocresol purple

is the pH indicator which turns purple colour in alkaline condition.

5.4.1.5 Ornithine decarboxylase: Pure cultures were aseptically inoculated into test tubes

containing sterile ornithine decarboxylase broth and incubated at 37oC in an incubator for

24-48 hrs. During the initial stages of incubation, glucose is fermented by the organisms

with acid production which results in the colour change of the pH indicator to yellow.

Further if the isolates which are able to decarboxylate the aminoacid ornithine will produce

an alkaline condition; as a result the colour of the medium will change from yellow to

purple.

5.4.1.6 Gelatin hydrolysis: Gelatin hydrolysis is shown by a test in which the organism is

aseptically inoculated into sterile nutrient gelatin tubes. The stab inoculated tubes were then

incubated at 37oC for about two days. Since gelatin at the concentration of 12% usually



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liquid at 37oC, liquefaction is tested at intervals by removing the nutrient gelatin cultures

from the incubator and holding them at 4oC for 30 min before reading the results.

5.4.1.7 Malonate utilization: Malonate broth is used for the differentiation of Enterobacter

and Escherichia. Organisms that simultaneously utilize sodium malonate as carbon source

and ammonium sulphate as nitrogen source produce an alkalinity due to formation of sodium

hydroxide. This change in pH is indicated by bromothymol blue indicator which change

colour from green to blue. The malonate broth tubes were incubated at 37oC for 18-48 hrs

before reading the results.

5.4.1.8 Esculin hydrolysis: Esculin agar is recommended for isolation and identification of

esculin hydrolyzing organisms. Esculin agar slants were aseptically inoculated with pure

cultures and incubated for 24-48 hrs at 37oC. Esculin hydrolysers hydrolyze the glycoside

esculin to esculitin and dextrose. Esculitin reacts with ferric citrate to form a dark brown to

black complex, which is considered as positive result.

5.4.1.9 Acetate utilization: Acetate differential agar is recommended for the differentiation

of Shigella species from members of the genus Escherichia. The differentiation is based on

the ability or failure of the test culture to utilize acetate. The acetate utilization will result in

the production of sodium acetate, which will change the pH into alkaline, which will be

indicated as blue colour by the bromothymol blue. Cultures aseptically inoculated into

acetate differential agar and observed for colour change after incubation at 37oC for 24-48

hrs.

5.4.1.10 ONPG test (o-nitrophenyl-β-D-galactopyranoside): Lactose fermentation depends

on the production of two enzymes-an inducible intracellular enzyme, β-galactosidase, which

hydrolyses lactose, and a permease which regulates the uptake of lactose into the cell.

Certain bacteria possess the β–galactosidase enzyme but not the permease. These potential

lactose fermenters may not produce acid at all in traditional peptone water media or may

take several days to do so. The ONPG test which determines the presence of the enzyme β-

galactosidase by utilizing o-nitophenyl- β-D-galactopyranoside. It is normally demonstrated

by acid production after the lactose is catabolised into glucose and galactose by the β-

galactosidase enzyme. Orthonitrophenyl β-D galactopyranose discs are immersed in 0.85%

NaCl solution and emulsified with the test organism. ONPG is chromogenic and when



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cleared off it produces a yellow solution. Inoculated tubes are read at 1, 2 and 24 hours of

incubation. A positive result is indicated by the development of yellow colour.

In addition to these tests, fermentation of various carbohydrate sources such as

glucose, adonitol, arabinose, cellobiose, dulcitol, inositol, lactose, mannitol, maltose,

raffinose, salicin, sorbitol, sucrose and xylose and gas production from glucose were also

determined by using phenol red broth base media (as detailed under section 4.4.1.j).

The Enterobacter strains were tested for indole production, acetoin production

(Voges Proskauer test), citrate utilization (Simmons) and ability to produce urease,

phenylalanine deaminase, nitrate reductase, DNase and lipase. The detailed procedures for

these tests are described in Chapter 4, section 4.4.1.b, 4.4.1.c, 4.4.1.d, 4.4.1.e, 4.4.1.m,

4.4.1.f, 4.4.1.o and 4.4.1.n respectively.

5.4.2 Differential characteristics of the species of the genus Alcaligenes

Species level characterization of Alcaligenes were carried out by the following

biochemical tests:

5.4.2.1 Acid from D-xylose in OF medium: Sterile oxidation fermentation (OF) medium

tubes were prepared with D-xylose as carbohydrate source and bromothymol blue as pH

indicator to differentiate breakdown of D-xylose. A small amount of pure culture is

inoculated into the medium by stabbing the butt and streaking the slant and incubated at

37oC for 24 hrs. Positive result was indicated by change in colour from green to yellow.

The Alcaligenes strains were also tested for nitrate reduction, gelatin hydrolysis and

the utilization of carbohydrates such as glucose, xylose, mannose and mannitol. These tests

were carried as detailed under section 4.4.1.f, 5.4.1.6 and 4.4.1.j.

5.4.3 Differentiation of Aeromonas species

Pure cultures of the genera Aeromonas were identified up to species level based on

their physiological and biochemical properties as per Bergey’s Manual of Determinative

Bacteriology, 9th edition (Holt et al., 2000).

Characteristics such as ability to grow at various NaCl concentrations such as 0%,

1% and 6% NaCl, gas production from glucose and ability to ferment various carbohydrates



132

such as glucose, adonitol, cellobiose, dulcitol, inositol, lactose, maltose, mannitol, raffinose,

rhamnose, salicin, sorbitol, sucrose and xylose were tested. Fermentation of the above

carbohydrate sources was carried out by using phenol red broth base media as detailed under

section 4.4.1.j.

Apart from the above tests other biochemical tests such as deamination and

decarboxylation of amino acids such as phenylalanine, lysine and ornithine, presence of

nitrate reductase, production of indole and acetoin, methyl red test, utilization of citrate and

malonate, and acid production from mucate were also carried out as detailed previously.

5.4.3.1 Acid production from mucate: Mucate broths containing mucic acid (is a

saccharolactic acid also called as tetrahydroxy adipic acid and act as a carbon source) and

bromothymol blue (as the pH indicator) were inoculated with pure cultures and were

incubated at 37oC for 24-48 hrs. If the isolates were able to ferment mucic acid will produce

acid and pH of the medium turn to acidic which will be indicated as yellow colour. A

negative test result is indicated by a blue/unchanged colour in this broth.

The Aeromonas strains were also tested for the presence of hydrolytic enzymes such

as, DNase, lipase, tyrosinase, esculinase and urease as detailed previously (in subsections of

4.4.1 and 5.4.1.8).

5.4.4 Differentiation of Vibrio species and biovars

Vibrio species were identified as per Bergey’s Manual of Determinative

Bacteriology, 9th edition (Holt et al., 2000) and also based on the dichotomous keys of

Noguerola and Blanch (2008) (Fig 1, 2, 3, 4, 5, 6, 7 and 8). The characters studied for the

identification of Vibrio were: arginine dihydrolase, lysine decarboxylase, ornithine

decarboxylase, growth at 0%, 6%, 8% and 10% NaCl, growth at 4oC, 35oC and 40oC,

utilization of citrate, gas production from glucose, indole production, nitrate reduction,

ONPG, oxidase, gelatinase, urease, amylase, lipase, tyrosinase, acid production from

carbohydrates such as glucose, cellobiose, inositol, lactose, mannitol, rhamnose, salicin,

sorbitol, sucrose, xylose and melibiose and the Voges Proskauer test. The procedure for

these tests are detailed previously and also in the Chapter 4 under subsection of 4.4.1.



133

Dichotomous key for the identification of Vibrio (Noguerola and Blanch, 2008).

Fig. 1

Fig.2

Fig. 3



134

Fig. 4



135

Fig. 5



136

Fig. 6

Fig. 7



137

Fig. 8



138

5.4.5 Differentiation of species of the genus Pseudomonas

Pure cultures of Pseudomonas were identified up to species level based on their

physiological and biochemical characters which include indole production, nitrate reduction,

lipase, gelatinase, tyrosinase, amylase, growth at 4oC, growth at 41oC and utilization of

various sugars such as glucose, inositol, trehalose, sucrose, xylose and rhamnose.

5.5 Results

In the present investigation an attempt has been made to study the diversity of Gram-

negative bacterial population in the water around Kumarakom region of Vembanadu Lake on

the basis of various morphological, biochemical and physiological characteristics of the

isolates. Isolates having the same colony morphology were often different with respect to

their physiological characteristics. Based on their character similarities, isolates were

classified into species.

5.5.1 Distribution of the members of the family Enterobacteriaceae in water samples

from Kumarakom region of Vembanadu lake

A total of 5 genera and 11 species of members of the family Enterobacteriaceae were

identified in water of the study area. The percentage incidence of different species of

Enterobacteriaceae isolated from water samples of the Kumarakom region of the Vembanadu

lake is given in Fig 5.1. Enterobacter cloaceae was found to be the dominant member of

Enterobacteriaceae in the study area. Where as, Proteus vulgaris and Enterobacter gergoviae

showed an equal percentage of incidences (11.54) in the study area. Three species of Klebsiella

such as Klebsiella terrigena, Klebsiella pneumoniae ssp. pneumoniae and Klebsiella

pneumoniae ssp. aerogenes were identified from water at Kumarakom region of Vembanadu

lake.

Enterobacter cloaceae isolated from four out of five sampling locations (Table 5.1).

Six species of Enterobacteriaceae were isolated from stations S1 and S5. Where as only two

species such as Enterobacter cloaceae and Proteus vulgaris were detected at station 4

(station near to Pathiramanal island).



139

30.77

11.54

11.54

15.38

7.69

3.85

3.85

3.85

3.853.85 3.85

Enterobacter cloacae Proteus vulgaris

Enterobacter gergoviae Enterobacter aerogenes

Escherichia coli Klebsiella terrigena

Ewingella americana Klebsiella pneumoniae ssp. pneumoniae

Enterobacter cancerogenus Enterobacter agglomerans

Klebsiella pneumoniae ssp. aerogenes

Fig 5.1: Percentage occurrence of different species of Enterobacteriaceae in the water samples of

Kumarakom region of Vembanadu Lake

Temporal variation of different members of the family Enterobacteriaceae isolated

from water in the Kumarakom lake is given in Table 5.2. More diverse species of

Enterobacteriaceae were detected during May-2008 followed by March-2008. Six species of

Enterobacteriaceae were identified during May-2008. Klebsiella terrigena detected in the

water samples collected during June-2008 and Enterobacter cancerogenus during May-

2008.



140

Table 5.1: Spatial distribution of various members of the family Enterobacteriaceae in water samples at Kumarakom region of Vembanadu lake

Members of the family Enterobacteriaceae

% occurrence in the water samples from different stations of Kumarakom lake

S1 S2 S3 S4 S5 Enterobacter cloacae 3.85 11.54 0 7.69 7.69

Proteus vulgaris 3.85 0 3.85 3.85 0

Enterobacter gergoviae 3.85 0 3.85 0 3.85

Enterobacter aerogenes 0 0 3.85 0 11.54

Escherichia coli 3.85 0 0 0 3.85

Klebsiella terrigena 0 3.85 0 0 0

Ewingella americana 0 3.85 0 0 0

Klebsiella pneumoniae ssp. pneumoniae 0 0 0 0 3.85

Enterobacter cancerogenus 3.85 0 0 0 0

Enterobacter agglomerans 0 0 0 0 3.85

Klebsiella pneumoniae ssp. aerogenes 3.85 0 0 0 0

Biochemical characteristics of Enterobacteriaceae isolated from water at Kumarakom

region of Vembanadu lake is given in Table 5.3. About 54% of isolates were able to produce

acetoin. Proteus vulgaris is the only H2S producer among the isolates. Seventy seven

percentage of Enterobacteiaceae strains were able to utilize acetate. The presence of β-

galactosidase was detected in 35% of Enterobacteriaceae isolates and 92% hydrolysed

esculin. Decarboxylation of aminoacids by Enterobacteriaceae revealed that 71% of

Enterobacteriaceae isolates were able to decarboxylate ornithine.



141

Table 5.2: Temporal distribution of various members of the family Enterobacteriaceae in water samples at Kumarakom region of Vembanadu lake during July-07 to June-08.

Members of the

family

Enterobacteriaceae

% of occurrence during different sampling months

Jul-2

007

Aug

-200

7

Sep

-200

7

Oct

-200

7

Nov

-200

7

Dec

-200

7

Jan-

2008

Feb

-200

8

Mar

-200

8

Apr

-200

8

May

-200

8

Jun-

2008

Enterobacter cloacae

3.85 0 0 3.85 0 0 3.85 0 3.85 3.85 7.69 3.85

Proteus vulgaris 0 0 0 0 0 3.85 0 0 0 0 7.69 0

Enterobacter gergoviae

0 3.85 0 0 0 0 0 3.85 3.85 0 0 0

Enterobacter aerogenes

3.85 0 0 0 0 3.85 0 0 3.85 0 3.85 0

Escherichia coli 0 0 0 0 0 0 0 0 3.85 3.85 0 0

Klebsiella terrigena

0 0 0 0 0 0 0 0 0 0 0 3.85

Ewingella americana

0 0 0 0 0 0 0 0 0 0 3.85 0

Klebsiella pneumoniae ssp. Pneumoniae

0 0 0 0 0 0 0 0 0 0 0 3.85

Enterobacter cancerogenus

0 0 0 0 0 0 0 0 0 0 3.85 0

Enterobacter agglomerans

0 0 0 0 0 0 0 0 0 3.85 0 0

Klebsiella pneumoniae ssp. aerogenes

0 0 0 0 0 0 0 0 0 0 3.85 0

Urease was detected in 38% of members of the family Enterobacteriaceae (Fig 5.2)

where as DNase was detected in 4% of strains. All Enterobacteriaceae isolates fermented

glucose and 96% of isolates fermented sucrose (Table 5.4). Only 19% of Enterobacteriaceae

sp. utilized adonitol as the source of carbohydrate.



142

Table 5.3: Biochemical characters of isolates of Enterobacteriacea from water at Kumarakom region of Vembanadu lake

Table 5.4: Carbohydrate fermentation ability of members of the family Enterobacteriaceae isolated from water at Kumarakom region of Vembanadu lake

Biochemical characters % of positives Carbohydrate

sources

% of positives

Methyl Red 42.31 Acid from glucose 100

Voges Proskauer 53.85 Acid from adonitol 19.23

Indole production 23.08 Acid from arabinose 26.92

H2S production 11.54 Acid from cellobiose 65.38

Ornithine decarboxylation 70.59 Acid from dulcitol 26.92

Phenylalanine deaminase 11.54 Acid from inositol 61.54

Arginine dihydrolase 00.00 Acid from lactose 76.92

Lysine decarboxylase 23.08 Acid from mannitol 80.77

Malonate utilization 46.15 Acid from maltose 73.08

Acetate utilization 76.92 Acid from raffinose 57.69

Nitrate reduction 100.0 Acid from salicin 88.46

Acid production from mucate 88.24 Acid from sorbitol 61.54

Citrate utilization 80.77 Acid from sucrose 96.15

Esculin hydrolysis 92.31 Acid from xylose 69.23

Gas production from D-glucose 61.54

0

5

10

15

20

25

30

35

40

45

Gelatinase DNase Urease Lipase β-galactosidase

% o

f po

siti

ves

hydrolytic enzymes

Fig 5.2: Hydrolytic enzyme production ability among the members of the family Enterobacteriaceae isolated from water at Kumarakom region of Vembanadu lake



143

5.5.2 Distribution of various species of Alcaligenes in water at Kumarakom region of

Vembanadu lake

In the present study biochemical characteristics of Alcaligenes isolated from water in

the Kumarakom region of the lake were investigated. Five species of Alcaligenes were

encountered based on their character similarities. The percentage incidence of various

Alcaligenes species is given in Table 5.5. Alcaligenes faecalis was found to be dominant and

about 16.67% of Alcaligenes strains are unidentified.

Table 5.5: Percentage incidence of different species of Alcaligenes isolated from water at Kumarakom region of Vembanadu lake

Alcaligenes species % incidence Alcaligenes faecalis 41.67 Alcaligenes xylosoxidans ssp. xylosoxidans 16.67 Alcaligenes latus 8.33 Alcaligenes paradoxus biovar II 8.33 Alcaligenes paradoxus biovar I 8.33 unidentified 16.67

The spatial distribution of different species of Alcaligenes isolated from water in the

Kumarakom lake is given in Fig 5.3. Alcaligenes faecalis was detected from all the four

stations (S1, S2, S4 and S5). More diverse Alcaligenes were identified in the water samples

from fifth (S5) station.

0

5

10

15

20

S1 S2 S3 S4 S5sampling sites

% in

cid

en

ce

Alcaligenes faecalis Alcaligenes xylosoxidans subsp. xylosoxidans

Alcaligenes latus Alcaligenes paradoxus biovarII

Alcaligenes paradoxus biovar I

Fig 5.3: Spatial distribution of Alcaligenes sp. isolated from water at Kumarakom region of Vembanadu lake

Species of Alcaligenes also revealed temporal variation. Alcaligenes faecalis showed

dominance during the month of July, while Alcaligenes xylosoxidans isolated during



144

September-2007 and April-2008. Temporal variation of Alcaligenes sp. in the Kumarakom

region of Vembanadu lake is given in the Fig 5.4.

Fig 5.4: Temporal distribution of Alcaligenes sp. in water at Kumarakom region of Vembanadu lake during July-07 to June-08.

Isolates of Alcaligenes showed distinct biochemical characters (Table 5.6). About

33% of Alcaligenes isolates reduced nitrate to nitrite. Glucose was found to be the most

preferred carbohydrate among the various carbohydrate sources studied. Biochemical

characters of Alcaligenes isolated from water in the Kumarakom lake is given in Table 5.6.

Table 5.6: Biochemical characteristics of Alcaligenes sp. isolated from water at Kumarakom region of Vembanadu lake

Biochemical characters % of positives

Utilization of glucose 33.33

Utilization of xylose 25.00

Utilization of mannose 08.33

Utilization of mannitol 16.67

Acid production from D-xylose in OF medium

27.27

Gelatin hydrolysis 33.33

Nitrate reduction 33.33



145

5.5.3 Distribution of Aeromonas species in water at Kumarakom region of Vembanadu

lake

Five species of Aeromonas were identified in the water of Kumarakom region of

Vembanadu lake. Their percentage incidence is given in the Fig 5.5. Most of the strains were

identified as Aeromonas eucrenophila (27.27%) and 18.18% of Aeromonas isolates were

identified as Aeromonas hydrophila. About 27% of Aeromonas strains were not identified up

to their species rank.

27.27

18.18

9.099.09

9.09

27.27

Aeromonas eucrenophila Aeromonas hydrophila

Aeromonas salmonicida subsp. smithia Aeromonas sorbia

Aeromonas caviae Unidentified

Fig 5.5: Percentage occurrence of different species of Aeromonas in the water at Kumarakom region of Vembanadu lake

The Aeromonas sp. were recovered from all the 5 sampling stations within the study

area (Fig 5.6). The presence of Aeromonas hydrophila were detected in the first (S1) and

second (S2) sampling stations. Aeromonas salmonicida subsp. smithia isolated from station

5 (S5) and Aeromonas eucrenophila from water samples of station 2 (S2).



146

05

1015202530

S1 S2 S3 S4 S5

sampling sites

% in

cid

ence

Aeromonas eucrenophila Aeromonas hydrophilaAeromonas salmonicida subsp. smithia Aeromonas sorbiaAeromonas caviae

Fig 5.6: Spatial distribution of different species of Aeromonas in the water at Kumarakom region of the Vembanadu lake

In the study area Aeromonas species were detected during the warmer times of the

year. The temporal variation of Aeromonas sp. in the water of the study area is given in the

Fig 5.7. Aeromonas eucrenophila were detected during March and April months.

0

2

4

6

8

10

12

14

16

18

20

Jul-0

7

Au

g-0

7

Se

p-0

7

Oct

-07

No

v-0

7

De

c-0

7

Jan

-08

Fe

b-0

8

Ma

r-0

8

Ap

r-0

8

Ma

y-0

8

Jun

-08

% in

cid

en

ce

Aeromonas eucrenophila Aeromonas hydrophila Aeromonas salmonicida subsp. Smithia

Aeromonas sorbia Aeromonas caviae

Fig 5.7: Temporal variation of Aeromonas species in water at Kumarakom region of Vembanadu lake during July-07 to June-08.

Hydrolytic enzyme activity of Aeromonas isolates from water of the study area is

given in the Fig 5.8. All the Aeromonas species studied in this investigation were able to

produce DNase. About 91% of isolates were able to hydrolyse Tween 80 as a lipid source.

While tyrosinase activity was detected in 50% of Aeromonas isolates only 27% hydrolysed

esculin.



147

0102030405060708090

100

DNase Lipase Tyrosinase Urease Esculinase

hydrolytic enzymes

% o

f po

sitiv

es

Fig 5.8: Hydrolytic enzymes in the isolates of Aeromonas from water at Kumarakom region of Vembanadu lake.

Table 5.7: Biochemical and physiological characters of Aeromonas isolates from water at Kumarakom region of Vembanadu lake

Table 5.8: Fermentation of carbohydrates by Aeromonas isolates from water at Kumarakom region of Vembanadu lake

Biochemical characters % of positives Carbohydrate source % of positives

Phenylalanine deaminase 27.27 D-glucose 100

Lysine decarboxylase 00.00 Adonitol 00.00

Ornithine decarboxylase 09.09 Cellobiose 09.09

Nitrate reductase 90.90 Dulcitol 09.09

Indole production 90.90 Inositol 09.09

Methyl red 18.18 Lactose 09.09

Acetoin production 63.63 Maltose 81.81

Citrate, Simmons 90.90 Mannitol 81.81

H2S production 00.00 Raffinose 09.09

Malonate utilization 09.09 Salicin 18.18

Mucate acid 40.00 Sorbitol 09.09

Growth at 0% NaCl 100 Sucrose 27.27

Growth at 1% NaCl 100 Xylose 00.00

Growth at 6% NaCl 30.00 Rhamnose 09.09



148

Aeromonas sp. were also studied for some of their physiological characters. Some of

the isolates (30%) were able to survive up to 6% of salt concentration (Table 5.7). Sixty

three percent of Aeromonas species were able to produce acetoin. About 91% of Aeromonas

were able to utilize citrate as the sole source of carbon, while 9% utilized malonate. About

27% of Aeromonas isolates were able to deaminate phenylalanine. Only 9% of the isolates

were able to decarboxylate the aminoacid ornithine.

The Aeromonas isolates were also tested for their ability to ferment one or other

source of fourteen different carbohydrates used in this study. All the isolates were able to

ferment glucose (Table 5.8). But none of the isolates were able to utilize xylose or adonitol.

Eighty one percent of Aeromonas isolates were fermented maltose or mannitol.

5.5.4 Distribution of Vibrio species and biovars in water at Kumarakom region of

Vembanadu lake

A total of 17 species of Vibrio were identified in the water samples of the study area,

based on their physiological and biochemical characters. The percentage incidence of Vibrio

sp. in the water of Kumarakom lake is given in the Fig 5.9. Vibrio coralliilyticus was the

most encounterd species, consisting of 17% of all the Vibrio’s isolated. It is followed by

Vibrio litoralis (13%). Ten percentages each of Vibrio agarivorans and Vibrio cholerae was

also identified in the water samples.

Table 5.9 represents the spatial distribution of Vibrio sp. in the study area. Vibrio

coralliilyticus was identified in all the five sampling sites. The presence of Vibrio

agarivorans was detected in the second (S2) and fourth (S4) sampling sites. Vibrio cholerae

was identified in the water samples from station 4 (S4) and station 5 (S5). Second sampling

site harbours maximum Vibrio species, about eight species of Vibrio were detected in this

site. Vibrio diversity was least at station 3 as it harbours only three species of Vibrio.

Temporal variation of Vibrio species in the water samples of Kumarakom lake is

given in the Table 5.10. More diverse species were obtained during June and Vibrio’s was

not observed during February. Six species of Vibrio were identified among the heterotrophs

isolated from water samples during June-2008.



149

0 5 10 15 20

Vibrio coralliilyticus

Vibrio litoralis

Vibrio agarivorans

Vibrio cholerae

Vibrio calviensis

Vibrio ponticus

Vibrio hepatarius

Vibrio pectenicida

Vibrio fisheri

Vibrio (colwellia) psychroerythrus

Vibrio pelagius

Vibrio alginolyticus

Vibrio natriegens

Vibrio logei

Vibrio vulnificus B2

Vibrio nigripulchritudo

Vibrio (Listonella) damsela

% incidence

Vib

rio

spe

cie

s

Fig 5.9: Percentage occurrence of Vibrio sp. in the water at Kumarakom region of Vembanadu lake



150

Table 5.9: Distribution of Vibrio sp. along different sampling sites around Kumarakom region of Vembanadu lake

Vibrio sp. % incidence in different sampling sites

S1 S2 S3 S4 S5

Vibrio coralliilyticus 3.33 3.33 3.33 3.33 3.33

Vibrio litoralis 3.33 3.33 0 3.33 3.33

Vibrio agarivorans 0 6.67 0 3.33 0

Vibrio cholerae 0 0 0 3.33 6.67

Vibrio calviensis 0 3.33 3.33 0 0

Vibrio ponticus 0 0 6.67 0 0

Vibrio hepatarius 0 0 0 3.33 0

Vibrio pectenicida 0 3.33 0 0 0

Vibrio fisheri 0 0 0 0 3.33

Vibrio (colwellia) psychroerythrus 3.33 0 0 0 0

Vibrio pelagius 0 3.33 0 0 0

Vibrio alginolyticus 0 0 0 0 3.33

Vibrio natriegens 0 0 0 0 3.33

Vibrio logei 0 3.33 0 0 0

Vibrio vulnificus B2 0 0 0 3.33 0

Vibrio nigripulchritudo 0 3.33 0 0 0

Vibrio (Listonella) damsela 3.33 0 0 0 0

Some of the physiological characters of Vibrio isolates from water in the

Kumarakom lake is given in Table 5.11. Nitrate reductase was detected in 63% of Vibrio

isolates from water in the Kumarakom lake. Fourty percent of isoates were able to survive

up to 8% of salt concentration and 25% of isolates up to 10% of salt concentration. About

54% of Vibrio isolates were able to grow up to 40oC and 90% utilized citrate as the source of

carbon.



151

Table 5.10: Temporal variation of Vibrio sp. in the water at Kumarakom region of Vembanadu lake during July-07 to June-08.

Vibrio sp.

Temporal variation of Vibrio sp. (% incidence) Ju

ne-

20

07

July

-20

06

Au

gu

st-2

006

Sep

tem

ber

-20

06

Oct

ob

er-2

006

No

vem

ber

-20

06

Dec

emb

er-2

00

6

Jan

uar

y-20

07

Feb

ruar

y-2

007

Mar

ch-2

007

Ap

ril-

20

07

May

-20

07

Vibrio coralliilyticus 0 3.33 3.33 3.33 0 0 3.33 0 0 0 0 3.33

Vibrio litoralis 3.33 3.33 3.33 0 0 0 0 0 0 3.33 0 0

Vibrio agarivorans 0 0 0 0 0 0 3.33 0 0 3.33 3.33 0

Vibrio cholerae 3.33 0 0 0 0 0 0 3.33 0 3.33 0 0

Vibrio calviensis 3.33 0 0 0 0 3.33 0 0 0 0 0 0

Vibrio ponticus 0 3.33 0 0 0 0 0 3.33 0 0 0 0

Vibrio hepatarius 0 0 0 0 0 0 0 0 0 0 0 3.33

Vibrio pectenicida 3.33 0 0 0 0 0 0 0 0 0 0 0

Vibrio fisheri 0 3.33 0 0 0 0 0 0 0 0 0 0

Vibrio (colwellia)

psychroerythrus

0 0 0 0 0 0 0 0 0 0 0 3.33

Vibrio pelagius 3.33 0 0 0 0 0 0 0 0 0 0 0

Vibrio alginolyticus 0 0 0 0 0 0 0 3.33 0 0 0 0

Vibrio natriegens 0 0 0 0 0 0 0 0 0 0 0 3.33

Vibrio logei 0 0 0 0 0 0 0 0 0 3.33 0 0

Vibrio vulnificus B2 0 0 0 0 3.33 0 0 0 0 0 0 0

Vibrio

nigripulchritudo

3.33 0 0 0 0 0 0 0 0 0 0 0

Vibrio (Listonella)

damsela

0 0 0 0 0 0 0 3.33 0 0 0 0



152

Table 5.11: Physiological and biochemical characters of Vibrio isolates from the water at Kumarakom region of Vembanadu lake

Physiological and

biochemical characters % of positives

Physiological and biochemical

characters % of positives

Growth at 0% NaCl 100.00 Arginine dihydrolysis 00.00

Growth at 6% NaCl 54.55 Decarboxylation of lysine 46.67

Growth at 8% NaCl 40.00 Decarboxylation of ornithine 43.33

Growth at 10% NaCl 25.00 Gas production from D-glucose 10.00

Growth at 4oC 60.71 Indole production 50.00

Growth at 40oC 53.57 Citrate utilization 90.00

Nitrate reduction 63.33 Production of acetoin 33.33

Hydrolytic enzyme production ability of Vibrio isolates from Kumarakom lake is

given in the Fig 5.10. Most of the isolates (93%) were able to hydrolyse starch. About 41%

of Vibrio in the present study hydrolysed tyrosine. While lipase was detected in 73% of

isolates, only 4% of Vibrio isolates exhibited urease activity.

0102030405060708090

100

Gel

atin

ase

Am

ylas

e

Lip

ase

Ure

ase

Tyr

osi

nas

e

β-

gal

acto

sid

ase

Hydrolytic enzymes

% o

f p

osi

tive

s

Fig 5.10: Hydrolytic enzyme production ability of the Vibrio isolates from water of Kumarakom region of Vembanadu lake.

Fermentation of various carbohydrate sources by the isolates of Vibrio is given in the

Fig 5.11. Glucose is the most preferred carbohydrate source, as 77% of strains fermented

glucose and lactose is the least preferred carbohydrate source (4%). About 46% of Vibrio

isolates fermented sucrose.



153

0

10

20

30

40

50

60

70

80

90

Aci

d p

rod

uctio

nfr

om

glu

cose

Aci

d p

rod

uctio

nfr

om

ce

llob

iose

Aci

d p

rod

uctio

nfr

om

inos

itol

Aci

d p

rod

uctio

nfr

om

lact

ose

Aci

d p

rod

uctio

nfr

om

ma

nnito

l

Aci

d p

rod

uctio

nfr

om

rh

am

no

se

Aci

d p

rod

uctio

nfr

om

sa

licin

Aci

d p

rod

uctio

nfr

om

so

rbito

l

Aci

d p

rod

uctio

nfr

om

su

cro

se

Aci

d p

rod

uctio

nfr

om

xyl

ose

Aci

d p

rod

uctio

nfr

om

me

libio

se

% o

f po

sitiv

es

Fig 5.11: Carbohydrate fermentation ability of isolates of Vibrio from water at Kumarakom region of Vembanadu lake

5.5.5 Distribution of Pseudomonas in water at Kumarakom region of Vembanadu lake

Two species of Pseudomonas were identified in water samples. They are

Pseudomonas putida and Pseudomonas stutzeri. Their percentage incidence is given in Fig

5.12. The Pseudomonas putida were identified in water samples from station 1 during

January-2008. But Pseudomonas stutzeri was identified in the first (S1) and fifth (S5)

sampling stations during May-2008 and January-2008 respectively.

33%

67%

Pseudomonas putida Pseudomonas stutzeri

Fig 5.12: Percentage incidence of various species of Pseudomonas from water at Kumarakom region of Vembanadu lake.



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All the Pseudomonas strains isolated from water in Kumarakom lake were able to

hydrolyse starch and produced indole. All of them were also able to survive up to 41oC.

About 67% showed nitrate reductase activity. Tyrosinase was detected in 33% of

Pseudomonas isolates (Table 5.12).

Table 5.12: Biochemical and physiological characters of isolates of Pseudomonas from

water at Kumarakom region of Vembanadu lake.

Biochemical and physiological characteristics

% positives Biochemical and

physiological characteristics % positives

Nitrate reduction 66.67 Indole production 100.00

Hydrolysis of Tween 80 00.00 Acid from D-glucose 00.00

Hydrolysis of tyrosine 33.33 Acid from inositol 00.00

Hydrolysis of starch 100.00 Acid from trehalose 00.00

Gelatin hydrolysis 00.00 Acid from sucrose 00.00

Growth at 4oC 00.00 Acid from xylose 00.00

Growth at 41oC 100.00 Acid from rhamnose 00.00

5.6 Discussion

The taxonomy and potential metabolic capabilities of culturable Gram-negative

bacteria from Kumarakom region of Vembanadu lake were studied. Assessment of the

potential metabolic capabilities provides a better picture of the ecological roles of the

organisms (Frette et al., 2004).

The release of effluents contributes greatly to the increase of bacterial population as

consequence of introduction of contaminant species and high quantity of organic residues

(Tam, 1998; Cotano and Villate, 2006). The contamination of aquatic environments by

effluents creates two kinds of public health problems: the first is related to the risks

associated with the primary contact (bathing), and the other related to consumption of fishes

and shellfishes caught from the polluted area. Microbiological examination forms the most

sensitive test for the detection of recent and potentially dangerous biological pollution,

thereby providing a useful assessment of water quality with high sensitivity and specificity.



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5.6.1 Diversity and significance of Enterobacteriaceae

In the present investigation 31% of Enterobacteriaceae were identified as

Enterobacter cloaceae. Enterobacter is a genus of straight gram-negative rods, lactose-

fermenting bacteria of the tribe Klebsielleae in the family Enterobacteriaceae, found chiefly

in the environment of water and soil but is common invaders of tissues in contaminated

wounds of animals and in opportunistic infections such as cystitis and pyelonephritis in

cattle. Only Enterobacter isolates that belong to the E. cloacae complex are of clinical

significance and are increasingly isolated as nosocomial pathogens (Hoffmann et al., 2005).

In surveillance studies, Enterobacter species are often not further classified beyond the

genus level probably because identification is difficult. It was reported that Enterobacter sp.

causes 7% of nosocomial infections in intensive care units in the USA (Streit et al., 2008). E.

aerogenes (syn. Klebsiella mobilis) is occasionally a cause of bovine mastitis, uterine

infections in mares and the mastitis-metritis-agalactia syndrome in sows. E. aerogenes is a

common agent of hospital-acquired infection. It exhibits a remarkable adaptive capability

and easily acquires resistance to β-lactam antibiotics during therapy (Thiolas et al., 2005).

In the present investigation about 4% of Enterobacteriaceae were identified as

Enterobacter cancerogenus, and it was detected in the first sampling station, ie. the station

near to Kumarakom Bird Sanctuary, one of the Kerala’s tourism icon. The Sanctuary acts as

a home for migratory birds and a resting place for water birds. Rivas et al. (2009) in their

study identified one strain closely related to Enterobacter cancerogenus in the water of the

Lake Martel, and they related the presence of this strain to the continuous flow of tourists in

this zone. Garazzino et al. (2005) reported that Enterobacter cancerogenus have been found

in human infections, and it is able to cause osteomyelitis in humans. The Enterobacter

strains have been recently proposed for water and wastewater treatments (Zhang and

Frankenberger, 2005). They reported that Enterobacter taylorae can enhance selenium

removal from river water. However it will be necessary to evaluate their potential

pathogenicity to man before further use in bioremediation studies (Rivas et al., 2009).

In the present study about 4% of Enterobacteriaceae were identified as the

Enterobacter agglomerans. Enterobacter agglomerans are also clinically important as it

cause septic arthritis (Flatauer and Khan, 1978). Enterobacter agglomerans are producers of

a complex of chitinolytic enzymes and can be used as an antagonist of many fungal



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phytopathogens (Chernin et al., 1996). Enterobacter is also known for nitrogen fixation in

soils. It is a free living nitrogen fixer that is distributed to the soil matrix via animal faeces.

These N2 fixing microbes grow in humid environments on leaf surfaces or in leaf sheaths

(phyllosphere), soil, and root surfaces. Potrikus and Breznak (1977) reported that

Enterobacter agglomerans are able to fix nitrogen.

The production of molecules with toxic activity by genetically transformed symbiont

bacteria of pest insects may serve as a powerful approach to biological control. The

symbiont Enterobacter gergoviae, isolated from the gut of the pink ball worm (PBW) has

been transformed to express Cyt IA, a cytolytic protein lethally toxic to mosquito and black

fly larvae as a model system (Kuzina et al., 2002). In the present study about 12% of

Enterobacteriaceae in the water at Kumarakom lake region were identified as Enterobacter

gergoviae.

In the present study 7.69% of members of the family Enterobacteriaceae were

identified as Escherichia coli. Mc Cormick et al. (1993) reported that Escherichia coli and

Salmonella enterica serovar typhimurium are among the most common causes of

gastroenteritis in humans. E. coli and other groups of coliforms may be present where there

has been faecal contamination originating from warm-blooded animals (Chao et al., 2003).

E. coli is recognized as a good indicator of faecal contamination. It is identified as the only

species in the coliform group which is found exclusively in the intestinal tract of human and

other warm-blooded animals and subsequently excreted in large numbers in feces,

approximately 109 per gram (Geldreich, 1983). Unlike other coliforms, E. coli which is

living as a commensal or parasite in the human or animal intestine, when voided in faeces, it

remains viable in the environment only for some days. Detection of E. coli in drinking water,

therefore, is taken as an evidence of recent pollution with human or animal faeces. E. coli is

the commonest cause of uncomplicated infections of the lower urinary tract and on the other

side some were causing severe infections of the upper tract (Hagberg et al., 1981). Though

the extended spectrum β–lactamase producing E. coli shows a chronic and invasive urinary

tract infections (Rodriguez et al., 2001), it is the most friendly microorganism for

biotechnologists as it is well studied and the most suitable host for rDNA technology.

Bacteria of the genus Klebsiella are a frequent cause of nosocomial infections (Horan

et al., 1988). Klebsiella sp. are ubiquitous in nature. Their nonclinical habitats encompass



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the gastrointestinal tract of mammals as well as environmental sources such as soil, surface

waters, and plants (Bagley, 1985). Environmental isolates have been described as being

indistinguishable from human clinical isolates with respect to their biochemical reactions

and virulence (Matsen et al., 1974). While the medical significance of Klebsiella obtained in

the natural environment is far from clear, such habitats are thought to be potential reservoirs

for the growth and spread of these bacteria which may colonize animals and humans (Knittel

et al., 1977). At present the genus Klebsiella is subdivided into five species. Clinically, the

most important species are K. pneumoniae and K. oxytoca, while K. ornithinolytica, K.

terrigena, and K. planticola are rarely isolated from human clinical specimens (Farmer et al.,

1985a; Podschun and Ullmann, 1998). K. planticola and K. terrigena are considered to be

environmental species, as reflected in their species designations. In contrast to K.

pneumoniae, neither species grows at elevated temperatures, such as at 44.5°C. Podschun et

al. (2001) reported a high percentage (53%) of surface water samples positive for Klebsiella

sp., the most common species being K. pneumoniae. K. pneumoniae and K. oxytoca,

although opportunistic pathogens (Cruickshank et al., 1975), are also widely distributed in

nature (Seidler et al., 1975) and can fix atmospheric nitrogen under some conditions

(Neilson and Sparrell, 1976).

In the present investigation Proteus vulgaris was identified in the water at

Kumarakom lake. P. vulgaris is a rod-shaped, Gram-negative bacterium that inhabits the

intestinal tracts of animals and humans, and is also found in soil and water, and can be

pathogenic. It is a chemoheterotroph in the Proteobacteria group. In people whose immune

systems are suppressed it can be an opportunistic pathogen, causing urinary tract infection,

pneumonia or septicemia. Although E. coli accounts for the largest percentage of cases of

uncomplicated cystitis, pyelonephritis, and prostatitis, Proteus ranks third as the cause of

these infections, particularly in hospital-acquired cases (Stamm, 1999). P. mirabilis accounts

for approximately 3% of nosocomial infections in the United States (CDC, 1996) and is

commonly isolated in clinical microbiology laboratories.

Proteus is unique, however, because it is highly motile and does not form regular

colonies. Instead, Proteus forms what are known as "swarming colonies" when plated on

non-inhibitory media. The most important member of this genus is considered to be P.

mirabilis, causative agent of wound and urinary tract infections. Fortunately, most strains of

P. mirabilis are sensitive to ampicillin and cephalosporins. Unlike its relative, P. vulgaris is



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not sensitive to these antibiotics. However, this organism is isolated less often in the

laboratory and usually only targets immunosuppressed individuals. P. mirabilis and P.

vulgaris can be differentiated by an indole test for which only P. vulgaris tests positive

(Gardner, 1995). Proteus vulgaris is able to produce indole from tryptophan, and gives a

positive reaction for methyl red test. These strains can hydrolyze urea rapidly, and have the

ability to degrade gelatin, lipids and DNA. They can deaminate phenylalanine. These species

also produce H2S on TSI agar slants (Collee et al., 1996).

5.6.2 Significance of Alcaligenes species

Alcaligenes sp. are small Gram negative rod shaped bacteria and most species are

found in marine environment has been known to be excellent hydrocarbon degraders

(Krooneman et al., 1996). Some members of this group like the A. dinifricans, A. odorans

and A. eutrophus are known to be excellent hydrocarbon degraders including the polycyclic

aromatic hydrocarbons (PAH) (Weissenfeis et al., 1990; Harayama et al., 1999). In the

present investigation about 42% of Alcaligenes were identified as A. faecalis and it is usually

considered a harmless saprophyte in the human intestinal tract. It seems to be well

established, however, that the organism can be pathogenic. Human infection in a number of

cases with the organism has been recorded and the clinical picture has varied, depending

upon the organ involved. Sherman et al. (1960) reported that A. faecalis was able to cause

infection in new born babies. Simmons et al. (1980) reported that A. faecalis was able to

cause an acute upper respiratory disease of turkeys (Turkey rhinotracheitis (coryza)).

A. faecalis is a heterotrophic nitrifying bacterium, which is commonly found in

wastewater treatment systems where it is used for the removal of nitrogen from wastewater

(Nishio et al., 1998; Kim et al., 2004; Joo et al., 2007). In the present study it was found that

33% of Alcaligenes from water in the Kumarakom lake have nitrate reductase. The phenol-

degrading ability of A. faecalis in wastewater sediments has also been documented (Tong et

al., 1998).

5.6.3 Diversity and significance of Aeromonas species

In the present investigation 5 species of Aeromonas were identified in the water at

Kumarakom region of Vembanadu lake. They are A. eucrenophila, A. hydrophila, A.

salmonicida, A. sorbia and A. caviae. Massa et al. (2001) also identified A. hydrophila, A.



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sorbia and A. caviae in the natural mineral water and well water, based on their reaction in

biochemical tests, i.e. acid from arabinose and salicin, aesculin hydrolysis and gas from

glucose. They reported that A. hydrophila and A. sorbia appear to be inherently more

virulent than those organisms classified as A. caviae. Le Chevallier et al. (1982) identified A.

sorbia strains from A. hydrophila by a positive Voges-Proskauer reaction, generally a

negative esculin hydrolysis test and negative salicin fermentation. Austin et al. (1989) have

shown that A. hydrophila. A. sobria and A. caviae comprise the most predominant clinical

isolates that are typically associated with fish.

In the present study about 18% of Aeromonas isolates were identified as A.

hydrophila. A. hydrophila is a Gram-negative ubiquitous aquatic bacterium, which has been

isolated from a wide range of water sources, such as river water, drinking water, as well as

water distribution pipe biofilms (Havelaar et al., 1990; Chauret et al., 2001; Lynch et al.,

2002; Bomo et al., 2004; Canals et al., 2006). A. hydrophila is able to produce cytotoxins

and enterotoxins that are often associated with acute gastroenteritis, as well as wound

infections in humans, peritonitis, meningitis, endocarditis, corneal ulcers, nosocomial

infections, urinary tract infections and less commonly associated with septicaemia of

immunocompromised patients (Janda, 1991; Kuhn et al., 1997; Janda and Abbott, 1998;

Fernandez et al., 2000). The potential health significance of Aeromonas sp. in aquatic

environments has been emphasized by several reports. These have shown that infections

caused by Aeromonas sp. are linked circumstantially with recreational and other activities in

aquatic environments (Fulghum et al., 1978; Joseph et al., 1979). The present study has been

carried out in the Kumarakom region of Vembanadu lake. The lake is used by over 1.6

million people, directly or indirectly, for agriculture, fishing, transportation and recreation.

The incidence of different species of Aeromonas in this study area may pose severe public

health risk.

In addition, A. hydrophila could also be pathogenic to fish, reptiles and amphibians,

causing haemorrhagic septicaemia (Fernandez et al., 2000). The pathogenicity of A.

hydrophila has been associated with toxins, proteases, outer membrane proteins,

lipopolysaccharides and flagella (Merino et al., 1996; Negueras et al., 2000; Rabaan et al.,

2001; Canals et al., 2006). Cahill (1990) correlated enterotoxin and cytotoxin production of

Aeromonas and certain biochemical tests, ie. haemolysis of rabbit erythrocytes, production

of acetoin (Voges-Proskauer (VP) reaction), lysine decarboxylation and sorbitol



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fermentation. Janda et al. (1983) found that only 42% of the 12 cytotoxic A. hydrophila

strains from pediatric patients were VP-positive. A strong correlation between cytotoxicity

and positive VP-reaction has been reported by Cumberbatch et al. (1979) and Burke et al.

(1983). In the present investigation it was found that 63.64% of isolates were able to produce

acetoin and 9.09% of Aeromonas strains were able to ferment sorbitol, but none of them

were able to decarboxylate lysine.

The study area is subjected to wide fluctuation in salinity on a seasonal basis. There

are reports on salinity stress and resultant out break of Epizootic Ulcerative Syndrome (EUS)

in fishes from this area. A. hydrophila was identified as a major pathogen in this EUS out

break. Also there are reports on distribution of A. hydrophila in aquatic environment and

fish. Hatha et al. (2005) reported prevalence of potentially virulent A. hydrophila in farm

raised fresh water fishes. Vivekanandhan et al. (2002) also reported the isolation of A.

hydrophila in marketed fish and prawn from the south India, in which 33.5% and 17.6%

prevalence of A. hydrophila, respectively was encountered.

The seasonal fluctuation of Aeromonas number might be caused by rainfalls and

pollution leakage together with adsorbed cells of these bacteria (Pianetti, 1998; Niewolak

and Opieka, 2000). These bacteria may survive for a long time in soil (Brandi et al., 1996)

where they are washed out to the river and may undergo proliferation if the water

temperature is adequate (Seidler et al., 1980). The temporal changes of their number in water

may be connected with a low temperature (Niewolak and Opieka, 2000). It is known from

literature (Kersters et al., 1995) that at water temperature below 15°C, besides the ability of

using trace amounts of organic carbon (Van Der and Hijnen, 1988), the speed of taking

nutrients by A. hydrophila does not stand the competition with psychrophilic heterotrophic

bacteria. Parveen et al. (1995) also reported that the seasonal peak of Aeromonas sp. was

observed in warmer months (April-May) in the surface waters. In the present investigation

various species of Aeromonas were detected in the warmer months of the year (ie. February,

March and April months).

In the present study it was found that all Aeromonas sp. isolated from water at

Kumarakom lake had DNase activity. Mejdi et al. (2011) reported that several virulence-

associated factors including adhesins, enterotoxins, hemolysins, proteases, DNases, lipases

and other extracellular enzymes, have been identified in Aeromonas sp. Some of these



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bacterial products cause tissue damage and aid in establishing an infection by overcoming

host defenses and by providing nutrients for bacterial proliferation (Agarwal et al., 1998).

About 91% of Aeromonas isolates from water samples of Kumarakom lake were

hydrolysed Tween 80. The production of maximum extracellular lipase by addition of

Tween 80 as best carbon source was observed by Handelsman and Shoham (1994) and

Neelambari et al. (2011). Lipases possess indigenous applications in recent technology.

Lipases have emerged as key enzymes in swiftly growing biotechnology and are used in

various industries like food, chemical, pharmaceutical, cosmetic, detergent production and

leather processing (Jaeger and Eggert, 2002) and especially in biodiesel production (Nelson

et al., 1996). In the last decade, lipases have gained importance to a certain extent over

proteases and amylases, especially in the area of organic synthesis. Aeromonas also could

prove to be useful in bioleaching processes, resulting in the minimisation of the negative

impact that certain substances, such as phosphorus (P) and potassium (K), have on the

economic functioning of the mine. Williams et al. (2008) reported the dominance of A.

hydrophila in the groundwater of the Sishen Iron-Ore Mine in South Africa.

5.6.4 Diversity and significance of Vibrio species

Fresh water environment represents a critical reservoir of Vibrio species (Caldini et

al., 1997). High densities of Vibrio sp. were reported from the inshore waters of Velar

estuary (Nair et al., 1980) from Cochin back waters (Chandrika, 1983), from the offshore

waters of Cochin backwater (Pradeep and Lakshmanperumalsamy, 1986; Lokabharathi et

al., 1987) and from Visakapattanam (Clark et al., 2003). In the present study 17 species of

Vibrio were identified in water at Kumarakom region of Vembanadu lake. It was also found

that Vibrio coralliilyticus was the most dominant species of Vibrio in the Kumarakom lake.

This is in sharp contrast to the results of similar investigations conducted on Atlantic waters

off Europe and America (Matte et al., 1994; Sunen et al., 1995; Barbieri et al., 1999) where

Vibrio alginolyticus was found to be the dominant resident Vibrio species.

Amirmozafari et al. (2005) reported that Vibrio fischeri, Vibrio harveyi and Vibrio

natriegens are much less halophilic and can not survive in waters with excess of 6% NaCl

concentrations. These 3 Vibrio species were most frequently encountered by them in

Gomishan coastal waters. They also reported that Vibrio alginolyticus is halophilic in nature



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and can tolerate up to 11‰ of saltiness. It is pathogenic for human and causes a variety of

extra-intestinal diseases such as soft-tissue infections, otitis and conjunctivitis (Matte et al.,

1994; Sunen et al., 1995; Mukherji et al., 2000; Penland et al., 2000). In the present study

also the presence of Vibrio fischeri, Vibrio natriegens and Vibrio alginolyticus were detected

and these are least abundant species in the Kumarakom lake.

In the present study 3.33% of Vibrio isolates were identified as Vibrio vulnificus.

This is in contrast to the report of Amirmozafari et al. (2005), who reported that Vibrio

vulnificus was the predominant species isolated from sea waters (41%). However, our study

area is not a true marine environment; saltiness varies considerably according to seasons.

The study area turns nearly fresh water lake during monsoon and turns saline during post and

pre-monsoon. Vibrio vulnificus is an autochthonous bacterium of estuarine and marine

waters of temperate and tropical climates (Oliver et al., 1982; Tamplin et al., 1982; Tamplin

et al., 1983), where seafoods and water can be vectors for transmission of V. vulnificus to

humans (Farmer et al., 1985b). V. vulnificus, V. carchariae and V. alginolyticus are widely

spread in the marine environment; both in closed seas, like the Mediterranean, and in oceans

(Matte et al., 1994; Rodrigues et al., 2001). Vibrio vulnificus has emerged as an important

pathogen responsible for septicemia and severe wound infections in immunocompromised

patients (Oliver, 1989). V. vulnificus is an estuarine bacterium commonly found in coastal

waters.

Among the aquatic microflora V. vulnificus, V. alginolyticus, V. cholerae and V.

parahaemolyticus are responsible for most infections by Vibrio in developing as well as in

developed countries (Faruque et al., 1998). They can cause most of the reported human

infections via ingestion or by direct skin penetration. Temperature and salinity are the two

major environmental factors that select the growth of these human pathogenic Vibrio (Kelly,

1982; Randa et al., 2004). Most Vibrio infections in temperate North America occur during

summer months (Oliver, 2005), primarily in the southern states when coastal water

temperature rise above 10°C.

Hatha et al. (2008) reported the presence of V. cholerae and V. parahaemolyticus in

various sampling stations of Kumarakom lake. In the present study also presence of V.

cholerae (10%) was detected in the water at Kumarakom lake. V. cholerae O1 and O139 will

be able to cause infection of the small intestine leading to cholera, a highly contagious



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disease, characterized by massive acute diarrhoea, vomiting and dehydration. Death occurs

in severe and untreated cases.

In the present investigation the presence of V. cholerae was identified at stations 4

and 5. In these stations the local inhabitants depends on the lake water for their livelihood

activities such as fishing, shellfish harvesting, transportation, recreation, agriculture etc. and

it also acts a destiny for backwater tourism. Effluents from houseboats are released

continuously to the water body. More diverse Vibrio species were identified in the second

sampling station, near to a hotel in Kumarakom. Flick (2007) reported that V.

parahaemolyticus, V. minicus and V. vulnificus are food-poisoning bacteria frequently

isolated from seawater and shellfish. It is also reported that V. cholerae is often transmitted

by water but fish or fish products that have been in contact with contaminated water or

faeces from infected persons also frequently serve as a source of infection (Kam et al., 1995;

Colwell, 1996; Rabbani and Greenough, 1999). The organically rich sedimentary substratum

of the Kumarakom estuarine region makes it a highly preferred and desirable habitat for

breeding of shrimps and other shell fishes. Also the traditional occupation of poor villagers

in this region is fisheries. Hence there is a chance of cross contamination of fishes and

shellfishes with Vibrio species from the polluted environment and can transmit disease

because they are filter-feeding organisms and can concentrate bacteria.

5.6.5 Significance of Pseudomonas

Microbes such as Pseudomonas sp, Enterobacter sp., Citrobacter etc. are able to

degrade organophosphate insecticides (Singh et al., 2004). Raghavan and Vivekanandan

(1999) illustrated successful bioremediation of oil in the open environment by the naturally

adapted P. putida which might serve as an important adjunct in oil spillage operations.

Kathiresan (2003) reported that mangrove soil is a good source of microbes, including

Pseudomonas sp., capable of degrading polythene and plastics. It is also reported that P.

stutzeri associated with Languncularia racemose was responsible for the fixation of

atmospheric nitrogen (Vazquez et al., 2000). P. aeruginosa is a Gram-negative rod-like

bacterium, which is ubiquitous in soil and water and commonly detected in great amounts in

sewage contaminated by humans and animals (Pellett et al., 1983). P. aeruginosa is an

opportunistic pathogen in humans and a major cause of nosocomial infection and are also



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able to cause urinary tract infections, particularly in immunocompromised patients (US Dept

of Health and Human Services, 2006).

The potential sanitary risk associated with the presence of these pathogenic Gram-

negative bacteria in the aquatic environment emphasizes the necessity of long-term

monitoring programs.

5.7 Conclusion

As the microorganisms are small, they are least known, and this gap in knowledge is

particularly apparent for bacteria and other small organisms. Current evidence suggests that

perhaps 1.5 million species of fungi exist yet only 5% are described. For bacteria there may

be 300,000 to 1 million species on earth yet only 3,100 bacteria are described in Bergey’s

Manuel, the treatise of described bacteria. A gram of typical soil contains about 1 billion

bacteria, but only 1% of those can be cultured. Similarly low fractions of microorganism

have been cultured from fresh water and ocean environments. Hence, most microbes remain

to be discovered.

The objective of the present study was to find out the diversity of culturable

heterotrophic bacteria. The Gram-negative forms showing good diversity with many species

belonging to different genera were identified. Spatial and temporal variations in their

distribution were noticed. While, some members of the genera Vibrio and Aeromonas are

pathogenic/potentially pathogenic and some may opportunistic pathogenic, many have

capabilities that could be exploited in potential biotechnological process. This is first report

of the diversity of culturable Gram-negative heterotrophic bacteria from the study area.

However, the difficulties in culture techniques remain major bottleneck in estimating the true

diversity.

New technologies, particularly in nucleic acid analysis, computer science, analytical

chemistry, and habitat sampling and characterization place the study of microbial diversity

on the cutting edge of science. The classification of bacteria has traditionally been a tedious

and often frustating task undertaken by every bacteriologist at some point in his/her carrier.

In this present investigation Gram-negative strains isolated from the wetland, were identified

up to the species level by the information provided by Bergey’s Manual of Determinative



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bacteriology. Vibrio was found to be the most dominant strain among Gram-negative

microbes in the study area. About 17 species of Vibrio, 11 species of Enterobacteriaceae, 5

species of Alcaligenes, 5 species of Aeromonas and 2 species of Pseudomonas were

identified in the present study.

Humans over the ages have been highly successful in applying processes carried out

by microorganism to solve problems in agriculture, food production, human health,

environmental quality and industry. Recently developed technologies in molecular biology

and genetics offer great promise for new opportunities to develop the potential of microbial

diversity.

Chapter 5 Diversity of Culturable Gram Negative Bacteria...

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