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Antagonistic activity of bacterial strains isolated from human producing biological control agents.

Microorganisms are not only the cause of infections; they can also produce organic substances that can cure infections (Jensen and Fencial, 2000) and various bioagents have been isolated from bacteria, fungus, and algae (Abdel-Fattah et al., 2011; Galal et al., 2011). Screening of bioactive compounds is based on some major factors like selection of a proper microorganism, isolation and culture methods and the detection and identification of their metabolites (Alwathnani and Perveen, 2012; Reddy et al., 2011).

Seven bacterial strains i.e., Steptococcus pyogenes, Staphylococcus epidermidis, Klebsiella pneumonia, Staphylococcus aurues, Serratia marcescens, E. coli and P. aeruginosa were isolated from different clinical samples of human. Most of the strains were catalase positive and indole negative (Awan et al., 2013). Antagonistic activity of human isolated pathogens was analysed through three agar disc diffusion systems such as phosphate buffer culture disc (PBCD) method, culture agar disc (CAD) method, and cell free supernatant disc (CFSD) method (Drummond and Waigh, 2000; Colle and Marr, 1989), zone of inhibition was measured in mm (Fig. 1).

Trivedi et al. (2008) and Trivedi and Sa (2008) demonstrated the antagonistic activity of Pseudomonas spp. against two phytopathogenic fungi, Fusarium oxysporium and Alternaria alternata. Similarly, in current research P. aureginosa results indicated that PBCD is much better method than rest of two methods. It has been analysed that P. aureginosa showed maximum activity against S. pyogenes, moderately high activity against S. epidermidis and moderately low against all other tested pathogens, respectively (Fig. 1A). Present results are consistent with Sindhu and Dadarwal (2001). The obtained results of TLC were found similar to that detected by Kumar et al. (2005) (Table 1).

In case of E. coli it was observed that S. aureus and S. epidermidis were significantly inhibited (15 [+ or -] 1 mm and 10 [+ or -] 1.73 mm) using CAD method. Similar results were observed by CFSD method of E. coli against S. aureus with 10 [+ or -] 1 mm zone of inhibition (Fig. 1). It has been proved that colicins produced by E. coli possess both antibacterial and antifungal activity (Nomura, 1967). Moderate activity was seen against P aureginosa through CAD method (8 [+ or -] 3.46 mm). This finding was consistent with Huang and Chang (1978). Significant results can be seen in case of S. epidermidis (maximum activity of 15 [+ or -] 1.52 mm against P aureginosa by CAD, and 10 [+ or -] 0 mm against S. pyogenes by PBCD) and S. aureus (maximum activity 12 [+ or -] 1.73 mm against P. aureginosa by CAD, followed by 15 [+ or -] 1.52 mm against S. epidermidis by PBCD, 15 [+ or -] 2 mm against K. pneumonia by CAD, and 10 [+ or -] 1 mm against E. coli by CAD) (Fig. 1). In case of K. pneumonia highest activity can be observed against S. pyogenes (10 [+ or -] 1.73 mm by CFSD), S. epidermidis (10 [+ or -] 1 mm by CAD), whereas moderate activity was indicated against E. coli (8 [+ or -] 1 mm by CAD) (Fig. 1). S.pyogenes showed significant activity against S. marcescens (14 [+ or -] 1 mm), S. aureus (14 [+ or -] 1.73 mm) by CFSD andE. coli (10 [+ or -] 1 mm) through CAD method, while reasonable but comparatively low activity against all other tested microbes (Fig. 1).A very low or no activity was observed by S. marcescens against all tested microbes via all three tested methods (Fig. 1).

In TLC analysis, different UV absorbing bands were observed under UV light (at 254 and 366 nm). Prominent coloured bands were observed by staining with anisaldehyde/[H.sub.2]S[O.sub.4]. The most promising diversity of coloured spots was seen in the crude extracts of three pathogens.

All the tested strains produced considerable amount of lactic acid and comparatively little amount of hydrogen peroxide (Table 2). This study revealed that the antagonistic activity perhaps indicated the potency of lactic acid and hydrogen peroxide production to inhibit the microbes.

References

Abdel-Fattah, G.M., El-Haddad, S.A., Hafez, E.E., Rashad, Y.M. 2011. Induction of defense responses in common bean plants by arbuscular mycorrhizal fungi. MicrobiologicalResearch, 166: 268-281.

Alwathnani, H.A., Perveen, K. 2012. Biological control of Fusarium wilt of tomato by antagonist fungi and cyanobacteria. African Journal of Biotechnology, 11: 1100-1105.

Awan, U.A., Andleeb, S., Kiyani, A., Zafar, A., Shafique, I., Riaz, N., Azhar, M.T., Uddin, H. 2013. Antibacterial screening of traditional herbal plants and standard antibiotics against some human bacterial pathogens. Pakistan Journal of Pharmacuetical Sciences, 26:1109-1116.

Colle, J.A., Marr, W. 1989. Cultivation of Bacteria. In: Practical Microbiology, Mackie & McCartney (ed.), pp. 121-140, 13th edition, Churchill Livingston, USA.

Drummond, A.J., Waigh, R.D. 2000. The development of microbiological methods for phytochemical screening. Recent Research Developments in Phytochemistry, 4: 143-152.

Galal, H.R.M., Salem, W.M., Nasr El-Deen, F. 2011. Biological control of some pathogenic fungi using marine algae extracts. Research Journal of Microbiology, 6: 645-657.

Huang, Tan-Chi, Chang, Miau-Chan. 1978. An antagonistic activity of Escherichia coli against a species of Pseudomonas. Botanical Bulletin of Academia Sinica, 19: 67-76.

Jensen, P., Fencial, W. 2000. Marine microorganisms and drug discovery current status and future potential. In: Drugs from the Sea, N. Fusetani (ed.), pp. 6-29, Karger, Basel, New York, USA.

Kumar, R.S., Ayyadura, N., Pandiaraja, P., Reddy, A.N., Venkates-Warlu, Y., Praakash, O., Sakthivel, N. 2005. Characterization of antifungal metabolite produced by new strain Pseudomonas aeruginosa PUPa3 that exhibits broad spectrum of antifungal activity and biofertilizing traits. Journal of Applied Microbiology, 98: 145-154.

Nomura, M. 1967. Colicins and related bacteriocins. Annual Review of Microbiology, 21: 257-280.

Reddy, N.G., Ramakrishna, D.P.N., Rajagopal, S.V. 2011. Optimization of culture conditions of Streptomyces rochei (MTCC 10109) for the production of antimicrobial metabolites. Egyptian Journal of Biology, 13: 21-29.

Sindhu, S.S., Dadarwal, K.R. 2001. Chitinolytic and cellulolytic Pseudomonas spp. Antagonistic to fungal pathogens enhances nodulation by Mesorhizobium spp. Cicer in chickpea. Microbiological Research, 156: 353-358.

Trivedi, P., Pandey, A., Palni, L.M.S. 2008. In vitro evaluation of antagonistic properties of Pseudomonas corrugata. Microbiological Research, 163: 329336.

Trivedi, P., Sa, T. 2008. Pseudomonas corrugata (NRRL B-30409) mutants increased phosphate solubilization, organic acid production and plant growth at lower temperatures. Current Microbiology, 56: 140-144.

Uzma Azeem Awan and Saiqa Andleeb *

Biotechnology Laboratory, Department of Zoology, Azad Jammu and Kashmir University, Muzaffarabad, Azad Kashmir, Pakistan

(received November 19, 2012; revised April 16, 2013; accepted April 19, 2013)

* Author for correspondence; E-mail: drsaiqa@gmail.com
Table 1. Thin layer chromatography of microbes by
using various solvent systems

Human microbes   UAW1         UAW2         UAW3          UAW4

Pseudomonas      S1 = 0.211   S1 = 0.961   no result     S1 = 0.608
  aeruginosa     S2 = 0.923   S2 = 0.980
Staphyloccocus   S1 = 0.063   no result    no result     S1 = 0.225
  epidermidis    S2 = 0.80
                 S3 = 0.87
Staphylococcus   S1 = 0.319   no result    S1 = 0.833    S1 = 0.394
  aureus         S2 = 0.382
                 S3 = 0.531
Klebsiella       no result    no result    S1 = 0.121    no result
  pneumonia      S2 = 0.292
                 S3 = 0.365
Escherichia      no result    S1 = 0.971   S1 = 0.0444   S1 = 0.4
  coli
Streptococcus    no result    S1 = 0.319   no result     no result
  pyogenes       S2 = 0.680
Serratia         S1 = 0.088   S1 = 0.782   no result     no result
  marcescens     S2 = 0.888

Human microbes   UAW5

Pseudomonas      no result
  aeruginosa
Staphyloccocus   no result
  epidermidis

Staphylococcus   S1 = 0.969
  aureus

Klebsiella       S1 = 0.6
  pneumonia

Escherichia      no result
  coli
Streptococcus    no result
  pyogenes
Serratia         S1 = 0.875
  marcescens

Note: spots on TLC indicated by S1, S2, S3 and solvent
systems were labeled as UAW 1; EtoAc: EthoH 9:1; UAW
2: EtoAc: MeoH 4:1; UAW3: EtoAc: Pet.ether 4:1; UAW 4:
EtoAc: Pet.ether 1:4; UAW5: Hex: Acetone 7:3.

Table 2. Estimation of biological control compounds
production by microbes

Bacterial isolates           Production of control agents (mg/mL)

                             Lactic acid   Hydrogen peroxide

Pseudomonas aeruginosa       54            0.535
Staphyloccocus epidermidis   54            1.605
Staphylococcus aureus        9             0.535
Klebsiella pneumonia         7.2           1.070
Escherichia coli             1.8           1.070
Streptococcus pyogenes       63            1.70
Serratia marcescens          2.7           1.070

Fig. 1. Antagonistic activity of human bacterial
pathogens with each other through phosphate
buffer culture disc method (PBCD),
culture agar disc method (CAD), and cell
free supernatant disc method (CFSD). A)
Pseudomonas aeruginosa; B) Staphyloccocus
epidermidis; C) Staphylococcus aureus;
D) Klebsiella pneumonia; E) Escherichia
coli; F) Streptococcus pyogenes; G) Serratia
marcescens.

A

Zone of inhibition (mm)

                             PBCD   CAD   CFSD

Streptococcus pyogenes       10     2     0
Staphylococcus epidermidis   3      8     4
Staphylococcus aureus        1      2     0
Serratia marcescens          4      0     2
Escherichia coli             3      0     0
Klebsiella                   1      0     2

B

Zone of inhibition (mm)

                             PBCD   CAD   CFSD

Streptococcus pyogenes       10     8     7
Staphylococcus aureus        7      15    10
Serratia marcescens          3      10    8
Escherichia coli             8      4     3
Klebsiella pneumonia         3      5     3
Pseudomonas aeruginosa       3      6     3

C

Zone of inhibition (mm)

                             PBCD   CAD   CFSD

Streptococcus pyogenes       7      4     8
Staphylococcus epidermidis   15     4     0
Serratia marcescens          2      9     6
Escherichia coli             3      15    6
Klebsiella pneumonia         9      10    0
Pseudomonas aeruginosa       2      12    8

D

Zone of inhibition (mm)

                             PBCD   CAD   CFSD

Streptococcus pyogenes       2      6     10
Staphylococcus epidermidis   3      10    5
Staphylococcus aerus         5      2     0
Serratia marcescens          3      4     2
Escherichia coli             4      8     6
Pseudomonas aeruginosa       2      3     2

E

Zone of inhibition (mm)

                             PBCD   CAD   CFSD

Streptococcus pyogenes       8      4     0
Staphylococcus epidermidis   7      10    5
Staphylococcus aerus         4      15    10
Serratia marcescens          3      4     5
Klebsiella pneumonia         3      7     5
Pseudomonas aeruginosa       2      8     6

F

Zone of inhibition (mm)

                             PBCD   CAD   CFSD

Staphylococcus epidermidis   0      2     5
Staphylococcus aerus         4      9     14
Serratia marcescens          4      10    14
Escherichia coli             3      1     10
Klebsiella pneumonia         1      1     4
Pseudomonas aeruginosa       8      6     1

G

Zone of inhibition (mm)

                             PBCD   CAD   CFSD

Streptococcus pyogenes       0      0     0
Staphylococcus epidermidis   0      0     1
Staphylococcus aerus         2      0     0
Escherichia coli             0      0     0
Klebsiella pneumonia         0      2     0
Pseudomonas aeruginosa       0      0     2

Note: Table made from bar graph.
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Title Annotation:Short Communication
Author:Awan, Uzma Azeem; Andleeb, Saiqa
Publication:Pakistan Journal of Scientific and Industrial Research Series B: Biological Sciences
Article Type:Report
Geographic Code:9PAKI
Date:Nov 1, 2013
Words:1661
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