Results
3.1. Actinomycetes isolation
Actinomycetes were isolated and the morphological appearance of isolates is shown in Figure 1.
Figure 1.
Morphological appearance of isolates.
3.2. Primary screening
Among the 31 actinomycetes isolated from water and sediments of Lake Tana, 13 isolates showed antibacterial activities against at least one of the tested bacteria. In perpendicular streak plate method, results revealed that the isolates, LT001 and LT009 exhibited broad spectrum activities against tested bacteria. The isolate LT002 showed potential activity against E. coli, P. aeruginosa, S. aureus and LT015 has shown a wide range zone of inhibition against E. coli, S. typhi and S. aureus. Isolates LT004 and LT007 was active only against P. aeruginosa. The isolates LT015 showed MIC (33 mm diameter) followed by LT009 (21 mm) and LT 002 (18 mm) against E. coli (Table 1).
Table 1
Zone of inhibition (mm) of the Isolates against tested bacteria using perpendicular streak method.
In agar overlay method, isolates of LT003, LT005 and LT010 was most effective against E. coli, P. aeruginosa, S. typhi and S. aureus. The isolate LT015 was active against E. coli, S. typhi, and S. aureus. The isolates of LT003, LT010 and LT005 showed maximum zone of inhibition i.e., 24 mm, 22 mm and 20mm respectively against P. aeruginosa. The isolate LT005 was observed with maximum zone of inhibition (30 mm) against S. typhi (Table 2).
Table 2
Primary screening of the isolates by using agar overlay method. Zone of inhibition (mm) against tested bacteria.
3.3. Secondary screening of crude extracts
The crude extracts prepared from 11 potential isolates by using solid state and submerged state fermentation methods was subjected to secondary screening by disc and agar well diffusion methods. In disc diffusion method, isolates LT001, LT002, LT004, LT005 and LT008 showed promising results against S. typhi and the results were statistically significant (df 5,12; F=62.90 P=3.44E-08). The isolates LT002 and LT004 was active against E. coli and the results was statistically significant within the tested isolates (df 2, 6; F=183.87; P= 4.14E-06). The inhibition zone of LT007 and LT010 was at the maximum of 11 mm against P. aeruginosa which was comparatively greater than positive control (10 mm) and the result was statistically significant within the crude extracts (df 4, 10; F=7.66; P=0.004 2) (Table 3). The isolates showing promising results against K. pneumonia was statistically significant (df 2, 6; F=6.81; P=0.028 5). Within the isolates tested against S. aureus showed statistically significant results (df 4, 10; F=275.40; P= 3.51E-10).
Table 3
Zone of inhibition (mm) in secondary screening of crude extracts (10 mg/mL) produced from solid state fermentation by using disc diffusion method.
In agar well diffusion method, maximum zone of inhibition (13 mm) was recorded from LT001 and LT008 against S. typhi and the results were statistically significant (df 3, 8; F=25.67; P=0.000 18). The inhibition zones of crude extracts from LT001 and LT002 were at the maximum of 12 mm and 10 mm respectively against E. coli and the results were statistically significant (df 2, 6; F=285.36; P=1.13E-06) (Table 4). The extracts of LT003, LT007 and LT009 showed statistically significant results against P. aeruginosa (df 3, 8; F=27.53; P=0.000 14).
Table 4
Zone of inhibition (mm) in secondary screening of crude extracts (10 mg/mL) produced from solid state fermentation by using well diffusion method.
The crude extracts obtained from submerged state fermentation, results revealed that LT003, LT006 and LT008 were active against S. typhi; LT003, LT005 and LT008 against E. coli; LT003 and LT009 against P. aeruginosa; LT005 and LT008 against K. pneumonia and LT003, LT005, LT008 and LT009 against S. aureus. The maximum inhibition zone of 11 mm was observed from LT008 against S. typhi which was comparatively similar to positive control (11 mm). The zone of inhibition was greater in all the tested extracts against E. coli compared to positive control. However, maximum inhibition zone of 15 mm was recorded from LT003 against E. coli which was comparatively greater than the positive control (10 mm) (Table 5). Within the isolates the results showed statistically significant (S. typhi, df 3.8; F=10.576; P=0.0037; E. coli, df 3,8; F=49.166; P=1.69E-05; P. aeruginosa, df 2, 8; F=8.25; P=0.0189; K. pneumonia, df 1, 4; F=9.375; P=0.0375 and S. aureus, df 4, 10; F=51.88; P=1.18E-06).
Table 5
Zone of inhibition (mm) in secondary screening of crude extracts (10 mg/mL) produced from submerged fermentation by using disc diffusion method.
The secondary screening of extracts produced from submerged state fermentation method was tested by agar well diffusion method and the results revealed that the zone of inhibition from LT008 was 14 mm; LT001 and LT003 was 13 mm against S. typhi. The activity of all tested extracts was comparatively greater than positive control (9 mm). Maximum zone of inhibition (17 mm) was recorded against E. coli from LT005. The zone of inhibition observed from LT005 and LT008 was superior to positive control (12 mm) (Table 6).
Table 6
Zone of inhibition (mm) in secondary screening of crude extracts (10 mg/mL) produced from submerged fermentation by using well diffusion method.
Within the isolates tested the results showed statistical significant (S. typhi, df 5, 12; F=19.739; P=2.06E-05; E. coli, df 4, 10; F=46.962: P=1.9E-06; P. aeruginosa, df 6, 14 F=45.92; P=2.04E-08; K. pneumonia, df 2, 6; F=2.654; P=0.149 and S. aureus, df 6,14; F=287.41; 7.8E-14). Among the crude extracts tested against individual bacterial strains, all showed statistically significant result (P<0.05) except K. pneumonia.
3.4. Determination of MIC and MBC
MIC of crude extracts ranged from 1.46 mg/mL to 2.52 mg/mL against Gram positive bacterium (S. aureus). In Gram negative bacterium such as E. coli MIC ranged from 1.84 mg/mL to 2.82 mg/mL. Among the 11 crude extracts tested the results of MBC ranged from 2.92 to 7.56 mg/mL against S. aureus and for E. coli, MBC ranged from 3.80 to 8.46 mg/mL (Table 7).
Table 7
MIC and MBC of the crude extracts (mg/mL).
3.5. Morphological characteristics of selected isolates
Results of morphological characteristics of the selected isolates revealed that the growth of the isolates was excellent in starch casein agar. The isolates of LT005, LT007 and LT015 showed excellent growth in starch casein agar and glycerol yeast extract. In mineral agar and starch casein agar growth was excellent for LT009. The aerial and substrate mycelium colour varied among the isolates such as LT004 was observed with blackish pigment and LT008 was observed with yellowish pigment (Table 8).
Table 8
Morphological characteristics of isolates.
3.6. Physiological and biochemical characteristics of isolates
Physiological and biochemical characteristics result indicates that all isolates showed the ability of starch and urea hydrolysis. The isolates from LT001 to LT005 were able to hydrolysis celatin; LT001 to LT003 and LT005 to LT008 were able to hydrolysis casein. The positive utilization of citrate was recorded in LT001, LT003, LT005, LT006 and LT009. The tested actinomycetes showed resistance capacity to grow in 5% concentration of sodium chloride. The optimum temperature for the growth of most isolates was between 25-30 °C and LT008 exceed up to 40 °C (Table 9).
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