Antagonistic activity of bacterial strains isolated from human producing biological control agents.
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.
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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: email@example.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|
|Date:||Nov 1, 2013|
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