Screening of Antifouling Compound Producing Marine Actinobacteria against Biofouling Bacteria Isolated from Poultries of Namakkal District, South India

 

R. Manikandan, R. Vijayakumar*

Research Department of Microbiology, Bharathidasan University Constituent College, Kurumbalur - 621107, Perambalur District, Tamilnadu, India

*Corresponding Author E-mail: rvijayakumar1979@gmail.com

 

Abstract:

Growth of biofouling microorganisms in surfaces of poultry, marine and other ecosystems is one of the major issues. Thus it is essential to control the growth of biofouling bacterial growth in surface of biological ecosystem. The present study has aimed to isolate novel antifouling compound from marine actinobacterial isolates. A total of 55 actinobacteria were isolated from Palk Strait coastal region (Bay of Bengal), Tamilnadu, India. All of them were preliminarily screened for their antifouling activity against six different biofouling bacterial (BFB) species isolated from poultry farms by cross streak plate method. Among them, 20 isolates possessed antibacterial activity against biofouling bacteria. From the 20 antifouling compound producers, 10 actinobacterial isolates were selected for further confirmation of antifouling compound production and their antifouling efficacies by shake flask culture method. Of them, one potential strain VS6 was found to be more active against all the six BFB. Thus, the result of the present study represents that the coastal areas of Tamil Nadu are rich in antifouling compound producing actinobacteria.

 

KEY WORDS: Palk Strait Coast, marine actinobacteria, poultry, antifouling activity.

 


 

Introduction:

Poultry is the livestock that contributes the largest share of animal protein for human diet. For this reason, it is one of the fastest growing agricultural sectors in India. This leads to significant raise in the production of broilers at the rate of 8 to 10% per annum with an annual turnover of 30,000 crores (Mehta and Nambiar, 2007). The advantages of low investment and the requirement of small area have directs to increase the number of poultry farms/shops and creates employment opportunities (Agblevor et al., 2010). This also leads to the generation of huge quantities of poultry wastes usually composed of broiler and layers, feathers, bones, blood, hatchery debris and dead birds.

 

The gases like ammonia and hydrogen sulfide causes sharp and pungent and rotten egg smell in poultry farms respectively. These wastes also pose serious environmental pollution problems through microbial infection, offensive odours, promotion of flies and rodent breeding (Adeoye Go et al., 1994). In particular, the odour/bad smell associated with urban chicken farming is a significant issue on not only on chickens but also on human life in the nearby area would be unbearable. In the poultry environment, microorganisms like bacteria and fungi are the major biofoul causatives (CDC, 1997). Bacteria which can grow as surface-associated aggregates on food contact-surfaces and equipments commonly referred to as biofilms (Chia et al. 2009). Usually, in most locations including natural, industrial, or clinical, biofouling bacteria are found in biofilms rather than in the planktonic state. Biofilms also provide important environmental reservoirs for pathogenic bacteria (Parsek and Singh, 2003), behind their survival in stressful environments, including food processing facilities and slaughterhouses (Chmielewski and Frank, 2003). In order to prevent the marine biofouling, already several primary biofouling agents namely chlorine dioxide (gas), sodium hypochlorite, glycolic acid, CO2 and thermal pasteurization and secondary biofouling agents namely chlorine dioxide (aqueous), H2O2, palimaleic acid, citric acid and ultrasound are reported. These biocides were very effective, but highly toxic to non-target organisms (Alzieu, 2000; Konstantinou and Albanis, 2004). Due to this adverse effect, International Maritime Organization (IMO) and Marine Environmental Protection Committee (MEPC) banned the usage of TBT or other substances containing tin as biocides in antifouling paints from January 2008 (Xu et al., 2010). Therefore it is necessary to develop alternative antifoulants and antibiofilm producers that are environmental friendly, as well as economically viable for poultry farms. From a biotechnological perspective, microorganisms particularly actinobacteria are an exploitable source of antifouling compounds, hence we focused our search on marine actinobacteria. Actinobacteria are a group of Gram-positive bacteria which form filamentous structure with asexual spores and have high guanine plus cytosine (G+C) content in their DNA. Diversity of actinobacteria in various natural and man-made ecosystems is well documented in recent years. They are primarily recognized as a source for high value metabolites such as antibiotics, antivirals, anticancers, enzymes and many recombinant products in which most of them are of terrestrial origin (Radhakrishnan and Balagurunathan, 2007; Vijayakumar et al., 2015). In the past 10 years, 659 marine bacterial compounds have been described with majority derived from actinobacteria (Williams, 2009). Recently, marine derived actinobacteria are also under exploitation especially for antibiotics, antifouling compounds and anticancer agents. Hence, the present investigation was designed for the isolation and screening of marine actinobacteria from Palk Strait coastal regions of Tamil Nadu for their biofouling bacterial inhibition potential against biofouling bacteria.

 

MATERIALS AND METHODS:

Collection of biofouling samples:

The biofouling samples were collected from the poultry interior area like cage, feeding bottle, tape connections and floor surface in and around Namakkal district, Tamilnadu, India. Samples were collected from six different regions namely P. Velur, Mohanur, Vasanthapuram, Pachudayam Patty, Nallipalayam and Thummankurichi. The samples were collected according to the standard microbiological procedures (Cappuccino and Sherman, 1993). In brief, the sterile cotton swabs were used to collect the samples from above said area of poultry and immediately transported to the laboratory for further analysis.

 

Isolation and Characterization of Biofouling Bacteria:

The samples were swabbed over nutrient agar plates in duplicate manner and all the plates were incubated at 37ᴼC for 24 h. After incubation, morphologically different bacterial colonies were purified using streak plate method and sub-cultured on nutrient agar slants (Bavya et al., 2011). A total of nine BFB were isolated and named as BB1-BB9.

 

Characterization and Identification of Biofouling Bacteria:

Characterization and identification of biofouling bacteria were carried out based on their growth pattern and colony characteristics on selective and differential media. In addition, Gram staining, motility determination, IMViC tests, and production of catalase, oxidase and urease were carried out according to the standard procedures described by Cappuccino (1993). Based on phenotypic (colonial and microscopical) properties, the biofouling bacteria were identified.

 

Isolation of Marine Actinobacteria:

Soil samples were collected from Palk Strait coastal regions including Point Calimere (Kodiyakarai), Vedaranyam, Muthupet mangrove forest, Adirampattinam, Mallipattinam and Manora of Tamilnadu, India. All the samples were labeled and brought to the laboratory. The soil samples were serially diluted and employed pure culture techniques on starch nitrate agar, glycerol asparagine agar, actinomycetes isolation medium, starch casein agar (SCA) for the isolation of actionbacteria. All the media were prepared with 50% sea water and added with griseofulvin at 50μg/ml to avoid fungal contaminations. The culture plates were incubated at 282ᴼC for 14 days. After incubation, the actinobacterial cultures were purified and stored in SCA slants.

 

Characterization of marine actinobacteria:

Purified isolates of actinobacteria were identified using morphological and cultural characteristics by the methods as described in the International Streptomyces Project (ISP) (Shirling and Gottlieb, 1966). The morphology of the spore bearing hyphae with the entire spore chain, the structure and arrangement of the spore chain with the substrate and aerial mycelium of the actinobacteria were examined using coverslip culture technique and identified (Williams et al., 1989). After growth, the slide cultures were examined under light microscope. Colour of spore mass was visually examined by using the colour chart (Pridham, 1965).

Preliminary screening of antifouling activity of actinobacteria:

The antifouling activity of the actinobacterial isolates were preliminarily screened by cross streak method (Egorov, 1985; Vijayakumar et al., 2012). Single streak of the actinobacteria was made on the surface of the modified nutrient agar medium and incubated at 282C. After observing a good ribbon like growth of the actinobacteria on the plates, the biofouling bacteria (BFB) namely BFB1-BFB9 were streaked at right angles to the original streak of actinobacteria and incubated at 37C, the inhibition zone was measured after 24-48 h. Based on the presence and absence of inhibition zone, the antifouling compound producing actinobacteria were selected for further study.

 

Secondary screening of antifouling activity of actinobacteria:

The actinobacteria with notable antifouling activity in preliminary screening were selected and inoculated into starch casein broth and shaken (at 250 rpm) at 282C for 7-10 days. After incubation, the staling substances were filtered through filter paper (No.1; Whatman, Maidstone, Kent, UK) and then through a Seitz filter (G5; Pall Corp, Port Washington, New York). An equal volume of different solvents namely acetone and ethanol was separately added to the cell-free culture filtrates and shaken for 2 h, followed by extraction of the antimicrobial compounds (Gandhimathi et al., 2008). Antimicrobial activity of actinobacteria was determined by using agar disc-diffusion method (Bavya et al., 2011). About 0.25 mg of crude extract was impregnated on filter paper disc (5 mm diameter) and placed on nutrient agar plates inoculated with selected BFB as lawn culture. All the plates were incubated at 28C for 24 h and observed for zone of inhibition. Diameter of inhibition zones was measured and recorded in millimeter. Streptomycin (20 g/ml) antibiotic or extract were used as positive and negative control respectively (Radhakrishnan et al., 2007; Dhanasekaran et al., 2008).

 

RESULTS AND DISCUSSION:

Poultry is recognized as one of the main roads by which zoonotic pathogens enter the chain of human food; therefore, much attention is given to their identification and elimination from the poultry farm (ACMF, 2005). One of the potential sources of poultry infection is water because with its help pathogens of salmonellosis and campylobacteriosis are transferred to the entire farm (Chaveerach et al., 2004; Zimmer et al., 2003). In the present study, the biofouling samples were collected from the floor surface, cage, feeding bottle and tape connections of poultry farms of Namakkal district, South India. All the collected biofouling samples were appeared as slimy in nature with white, cream and yellow coloured. Total culturable BFB from different poultry farms were estimated and recorded in table 1.

 


 

 

Table 1. Morphological properties of biofouling bacteria

Culture code

Colony morphology of the bacteria

Shape

Elevation

Edge

Color

Surface

Colony size (mm)

Bacillus sp. BFB1

Circular

Flat

Entire

Colorless

Smooth

1.9

Bacillus sp. BFB2

Circular

Flat

Entire

Colorless

Smooth

1.5

Bacillus sp. BFB3

Circular

Convex

Entire

White

Smooth

2

Staphylococcus sp. BFB4

Circular

Convex

Entire

Yellow

Smooth

1.2

Micrococcus sp. BFB5

Circular

Convex

Entire

White

Smooth

1

Streptococcus sp. BFB6

Circular

Flat

Entire

Cream

Smooth

1.7

BFB = Biofouling bacteria

 

Table 1. Conti................

Culture code

Microscopy

Growth on NA

Growth on MSA

Starch Hydrolysis

Catalase

IMViC

Growth at 6.5% NaCl

Gram staining

Motility

ES

Bacillus sp. BFB1

G+ve rod

+

+

Colorless

NG

+

+

- - + +

+

Bacillus sp. BFB2

G+ve rod

+

+

Colorless

NG

-

-

- - - +

-

Bacillus sp. BFB3

G+ve rod

+

+

White

NG

+

+

- - - -

+

Staphylococcus sp. BFB4

G+ve coccus

-

-

White

Yellow

ND

+

ND

+

Micrococcus sp. BFB5

G+ve coccus

-

-

Yellow

ND

ND

+

ND

ND

Streptococcus sp. BFB6

G+ve coccus

-

-

White

ND

ND

-

ND

ND

BFB = Biofouling bacteria ; + = positive, - = negative; G+ve = Gram positive; G-ve = Gram negative; ES = endospore staining; NA = nutrient agar; MSA = mannitol salt agar; IMVic = indole, methyl red, Vogus-Proskauer and citrate utilization test; ND = not determined

 


Totally, 6 BFB isolates were isolated from the biofouling samples. All the 6 BFB were identified at genus level. Three bacterial isolates belonged to the genus Bacillus spp. (BFB1, BFB2 and BFB3), each one isolate to Staphylococcus sp. (BB4), Micrococcus sp. (BB5) and Streptococcus sp. (BB6) (Table 1). The BFB have been already reported from different habitats (Sillankorva et al. 2008). Correspondingly, Gopikrishnan et al. (2013) isolated and reported the biofouling bacteria namely Bacillus, Aeromonas, Micrococcus, Alcaligenes, Lactobacillus, Staphylococcus, Pseudomonas and Kurthia from Parangipettai, Nagapattinam and Ennore coastal areas. Also, Bacillus sp. (BB11), Serratia sp. (BB13) and Alteromonas sp. (BB14) was reported from biofouling samples of Parangipettai coastal area by Bavya et al. (2011). Thus the present and earlier studies it has been reported that Bacillus species was frequently isolated and reported from many biofouling samples than other BFB. The present study has isolated a total of 1,394 marine actinobacterial colonies from Palk Strait coastal regions of Bay of Bengal on 4 different culture media namely starch nitrate agar, glycerol asparagine agar, actinomycetes isolation medium, SCA. From these 1,394 colonies, 55 were morphologically distinct isolates (MDI) with each other based on their colony size, nature, colour of aerial spore mass and reverse side colour, formation of aerial and substrate mycelia, spores/sporangia on aerial and substrate mycelia. Of the six different sampling stations, Muthupet mangrove soil contributed maximum (744 CFU; 50.9% of MDI) actinobacterial population, followed by Mallipattinam (170 CFU; 7.2% MDI), Adirampattinam (160 CFU; 7.2% MDI), Point Calimere (137 CFU; 12.7% MDI), Manora (127 CFU; 10.9% MDI) and Vedaranyam saltpan (56 CFU; 10.9% MDI). Among the 55 isolates of actinobacteria, Streptomyces (n=21) was dominant genera, followed by Nocardiopsis (n=4), Nocardia (n=2), Actinoplanes (n=3), Actinobispora (n=2), Actinopolyspora (n=2), Actinokineospora (n=1), Saccaropolyspora (n=6), Streptosporangium (n=2), Actinosynnema (n=1), Catellospora (n=2), Micromonospora (n=1), Streptoverticillium (n=1), Micromonospora (n=1), Dactylosporangium (n=1), Actinomadura (n=4) and Kitasatospora (n=1). Out of 55 isolates, 30 isolates showed their growth within three days of incubation and rest of isolates showed their growth after five days only. Based on the colour of aerial spore mass of the isolates, white colour series are dominant than grey and dove colour series. Correspondingly, the diversity of actinobacteria from different marine environments were studied and recorded by many workers, from marine sediments of east coast of India (Sudha and Selvam, 2012); deep sea (10,898 m) sediment of Mariana Trench (Pathom-aree et al., 2006); marine sediments of Bay of Bengal (Thirumurugan and Vijayakumar, 2013); mangrove sediments (Sathiyaseelan and Stella, 2011); saltpan and seashore (Vijayakumar et al., 2007). In addition, like the present study many workers (Vijayakumar et al., 2007; Zerizer et al., 2013) reported that among the actinobacteria, Streptomyces is the dominant genera than other actinobacteria. In order to obtain effective antifouling agents, antifouling activity of all the 55 marine actinobacteria was screened by cross streak plate method against six BFB namely Bacillus spp. (n=3), Micrococcus sp. (n=1), Staphylococcus sp. (n=1), Streptococcus sp. (n=1). Among 55 actinobacteria, 20 (36.36%) isolates of actinobacteria possessed antifouling activity against at least any one BFB. From the 20 antifouling actinobacteria, the isolates Streptomyces sp. VS6, Catellospora sp. MS3, Streptoverticillium sp. MUS2 and Streptomyces sp. MUS12 were exhibited noticeable activity against all the tested biofouling bacteria (Table 2).


 

Table 2. Primary screening of antifouling activity of marine actinobacteria

S. No.

Isolate code

Zone of inhibition (mm)

Bacillus sp. BFB1

Bacillus sp. BFB2

Bacillus sp. BFB3

Staphylococcus sp. BFB4

Micrococcus sp. BFB5

Streptococcus sp. BFB6

1

KS1

-

-

5

-

-

-

2

KS2

-

-

-

-

-

2

3

KS6

-

-

-

-

-

1

4

VS4

-

-

4

-

3

3

5

VS5

-

-

-

-

-

3

6

VS6

22

24

25

21

23

21

7

AS1

-

-

-

46

37

52

8

MPS2

-

-

-

-

4

4

9

MS2

-

21

22

27

22

25

10

MS3

3

4

45

51

62

47

11

MS5

-

1

15

17

2

2

12

MS6

2

-

3

-

-

2

13

MUS2

2

25

1

17

3

4

14

MUS5

2

-

4

-

2

3

15

MUS6

2

-

2

1

1

-

16

MUS10

-

-

4

-

-

-

17

MUS11

-

-

2

-

3

-

18

MUS12

2

2

2

14

4

4

19

MUS13

28

-

-

4

-

-

20

MUS24

-

-

-

3

4

-

- = No activity; BFB = biofouling bacteria


Further, the antimicrobial efficacies of the four dominant antifouling compound producing actinobacteria were tested. Among them, ethanol extract of the isolate Streptomyces sp. VS6 showed maximum (30 mm) activity against Streptococcus sp. BFB6, followed by 29 mm against Bacillus sp. BFB3, 15 mm against Bacillus sp. BFB1, Bacillus sp. BFB2 and Micrococcus sp. BFB5 and 13 mm against Staphylococcus sp. BFB4 at 400g/disc (Fig. 1). Whereas, other concentrations of the ethanol extract and all the concentrations of acetone extract of the Streptomyces sp. VS6 showed moderate activity at all the concentrations to all BFB (Fig. 2). Similarly, Gopikrishnan et al. (2013) reported the maximum antifouling activity of marine actinobacterium PM33 against Micrococcus sp. (M50). Likewise Bavya et al. (20) reported ethyl acetate extract of actinobacterium strain R1 showed maximum activity against Bacillus sp. (BB11), Serratia sp. (BB13) and Alteromonas sp. (BB14).

 


 

 

 

Fig. 1. Antifouling efficacy of Streptomyces sp. VS6 ethanol extract

 

 

Fig. 2. Antifouling efficacy of Streptomyces sp. VS6 acetone extract

 

 


CONCLUSION:

The present study revealed that the marine actinobacteria of Palk Strait region are potential source for the development of novel antifouling compounds against biofouling bacteria isolated from poultries. To confirm this, further studies are needed with respect to surface attachment and biofilm forming capability of biofouling bacteria, and the characterization of the antifouling compound of potential isolate Streptomyces sp. VS6 to identify the chemical nature of the active compound.

 

ACKNOWLEDGEMENTS:

This work was supported by grants from the Tamilnadu State Council for Science and Technology, Chennai, India (Project Ref. No. TNSCST/S&T Projects/VR/ES/2013-2014/468 dated 16.04.2014).

 

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Received on 31.03.2016 Modified on 23.04.2016

Accepted on 30.04.2016 A&V Publications All right reserved

Research J. Science and Tech. 2016; 8(2):83-89

DOI: 10.5958/2349-2988.2016.00011.5