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2 authors:
Mohammed Fadhil
Mohammad J. Al-Jassani
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ISSN: 2320-2246
ABSTRACT
Fungi were the first producers of antibiotics since 1928. Penicillium is one of the most important antibiotic
producers that need to be thoroughly investigated for antibiotic production. The effect of different pH,
temperature and duration for production of the antimicrobial metabolite were investigated using four natural
liquid media. The mold isolate (P1ROR) was identified morphologically and by sequencing the ITS region as a
novel Penicillium sp. strain. The mold isolate was found active in producing an antibiotic against both pathogenic
penicillin resistant bacteria and pathogenic molds. The potato infusion with added table sugar was the best and
economic liquid medium for antimicrobial metabolite production, while the optimum fermentation conditions
were pH 6.5 at 30C for 5 days. The produced antimicrobial metabolite with its valuable properties is highly
needed especially with the emergence of resistant strains and need to put in practice.
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Composition
1% Table sugar + potato infusion
1% Glucose + potato infusion
1% Table sugar + bran infusion
1% Glucose + bran infusion
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Sequence
Tm (C)
ITS1
5` TCCGTAGGTGAACCTGCGG 3`
61
ITS4
5` TCCTCCGCTTATTGATATGC 3`
55.3
2.4 Sequencing
The purified PCR product of the mold isolate was
subjected to the sequencing reaction following the
manufacturers recommendation using the BigDye
Terminator v3.1 sequencing reaction Kit (Applied
Biosystems, Foster City, CA, USA). The reaction mix was
set up in 10l final reaction (Table 4). ITS4 primer was
used for partial ITS region sequencing of the selected
mold isolate. The reaction tubes then transferred to the
BioRad C1000 thermal cycler (BioRad Laboratories,
Inc.). The sequencing reaction and cycle conditions
were optimized and as the following: Pre-denaturation
at 98C for 5min; 30 cycles of (denaturation at 96C for
20sec., annealing at 56C for10 sec., extension at 60C
for 4min.); final extension at 60C for 5min then hold at
4C. The BigDye terminator ready reaction mix was
added after the pre-denaturation step.
The sequencing product was purified using Sephadex
G-50 (Sigma-Aldrich, USA) with a spin column. The
collected DNA was transferred to the ABI prism 3130XL
genetic analyzer (Applied Biosystems, Foster City, CA,
USA) for sequence recording.
Volume (l)
12.925
2.5
1
0.2
0.5
0.5
0.125
2
0.25
5
25
Final concentration
1X
1 mM
0.2 mM
0.2 mM
0.2 mM
0.03 U/ml
1M
0.1 mg/ml
0.8-1.6
Volume (l)
4
1
1
4
10
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Mohammed F. Al-Jawad et al. / Curr Res Microbiol Biotechnol. 2015, 3(5): 725-732
Figure 1: The primary screening of the isolate P1ROR against S. aureus (left) andS. pyogenes (right). A: The isolate P1ROR with
the pathogenic bacteria. B: Control (without producer).
Figure 2: The secondary screening of the isolate P1ROR against M. canis (left) and T. mentagrophytes (right). A: The isolate
P1ROR with the pathogenic fungi. B: Control (without producer).
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Mohammed F. Al-Jawad et al. / Curr Res Microbiol Biotechnol. 2015, 3(5): 725-732
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Mohammed F. Al-Jawad et al. / Curr Res Microbiol Biotechnol. 2015, 3(5): 725-732
zone 3.5cm for S. aureus and 2.8cm for M. canis, but the
activity decreased with high neutral conditions and no
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4. CONCLUSION
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Mohammed F. Al-Jawad et al. / Curr Res Microbiol Biotechnol. 2015, 3(5): 725-732
Figure 9: Neighbor-joining tree of the P1ROR isolate. The species from which strains were isolated, strain number and GenBank
accession numbers are mentioned.
5. REFERENCES
1.
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4.
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