NewBioWorld A
Journal of Alumni Association of Biotechnology (2020) 2(1):33-37
RESEARCH
ARTICLE
Antibiotic Efficacy Assessment of Certain Species of Penicillium fungi
Nagendra Kumar Chandrawanshi*,
KL Tiwari, SK Jadhav
School of Studies in Biotechnology, Pt. Ravishankar
Shukla University, Raipur, Chhattisgarh, India
*Corresponding Author Email- chandrawanshi11@gmail.com
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ARTICLE INFORMATION
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ABSTRACT
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Article history:
Received
05 November 2019
Received in revised form
12 December 2019
Accepted
Keywords:
Penicillium species; Antimicrobial
activity; Broth dilution assay;
Bacteria and Inhibition
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This study aimed to evaluate the antimicrobial activity
of Penicillium species
on soil-isolated gram-positive coccus bacteria. The antimicrobial efficacy
of Penicillium species
filtrate extracts were determined via disc diffusion and broth dilution
assay. A kill curve study was conducted with treated and untreated bacterial
populations. There were P.
oxalicum, and P.
purpurogenum showed the maximum zone in solid surface culture
methods, followed by other spices with fewer antibiotic properties. The kill
curve study has shown an exposure-dependent inhibition for bacterial
populations. With the increase in the exposure periods, microbial growth was
significantly reduced. In the broth dilution assay methods, among all
treated Penicillium species,
only P. oxalicum and P. funiculosum species were
shown potent antibiotic properties; other species had given the very least
activity. The study interpreted that screening of the suitable strain of
microbes for very essentially for industrially important bioactive compounds
scale up at industrial set up.
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Introduction
Fungi play
a significant role in soil ecosystems, along with bacteria, protists, small
invertebrates, and plants, through complex trophic interactions. Most soil
fungi are regarded as saprobes, decomposing organic matter and contributing to
nutrient cycling, while several species form mycorrhizal associations with
plants or are plant pathogens. Also recognized as prolific secondary metabolite
producers, fungi have provided several bioactive compounds and chemical models
currently used as pharmaceuticals, and soils are traditionally the primary
source of fungal genetic resources for bioprospection programs (Adrio &
Demain, 2003). Penicillium is a large anamorphic
(asexual state) ascomycetous fungal genus with widespread occurrence in most
terrestrial environments. This genus comprises more than 200 described species,
and many are common soil inhabitants, as well as food-borne contaminants or
food ingredients used in the preparation of cheese and sausages. Penicillium species
produce a much-diversified array of active secondary metabolites, including
antibacterial, antifungal substances, immunosuppressants, cholesterol-lowering
agents, and potent mycotoxins (Lucas et al., 2007). Thousands of Penicillium isolates
have probably been screened in bioprospecting programs since the discovery of
penicillin (Petit et al., 2009). The synthesis of large numbers of antibiotics
over the past three decades caused complacency about the threat of antibiotic
resistance. Drug resistance is frequently encountered in hospital-acquired
pathogens, usually critically ill or immune-suppressed patients. The resistance
of microorganisms to low-cost antibiotics also significantly increases the
country's healthcare spending (Friedman et al., 2016). Thus, the pharmaceutical
industry and academic institutions invest vast resources to produce safe and
effective antimicrobial drugs (Strobel & Daisy, 2003). The members of the
genus Penicillium are well known for the production of
antibiotics. P. purpurogenum has been isolated from
various substrates, particularly soil, and wood. They are known for producing
polyketide red pigments, natural colorants in the food industry (Padmapriya et
al., 2015). However, their antimicrobial activity needs to be better
characterized. The reports on endophytic strains of P. purpurogenum are
also only few available. However, previous penicillin production investigators
have yet to develop a highly effective medium successfully. For decades,
penicillin yields have been increased through the development of better
production strains by classical mutagenesis procedures and optimization of the
growth conditions. Penicillin biosynthesis is regulated by environmental
factors such as the medium's phosphate, carbon, nitrogen, and oxygen content
(Feng et al., 1994). Thus, this study aimed to evaluate the
antimicrobial activity of various species of Penicillium against
soil-isolated cocci bacteria (cocci morphology). The fungal bioactive
constituents of mycelium filtrate of antibacterial activity were characterized
via gel diffusion and broth assay analysis.
Materials and Methods
Microorganisms and maintenance of
culture
DOI: 10.52228/NBW-JAAB.2020-2-1-7
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The identified microorganisms were obtained from the School of Studies in
Biotechnology, Pt. Ravishankar Shukla University, Raipur. The microorganisms
used in this study were ten fungi (Penicillium citrinum, P. notatum, P.
oxalicum, P. rubrum, P. frequent, P. rugulosum, P. chrysogenum, P. variabile, P.
purpurogenum, and P. funiculosum. The soil isolated
gram-positive cocci, biochemically characterized bacteria used for the
antimicrobial assay. The test microorganisms were sub-cultured on nutrient agar
before use every two weeks to maintain their viability. The microbial inoculums
were prepared by transferring a loopful of microbial colonies into a universal
bottle of sterile distilled water. The turbidity of the suspension was adjusted
to approximately 108 colony-forming units (CFU)/mL (Yenn et al., 2017).
Culture medium
Potato
dextrose agar (PDA) [200g/L potato extract, 20 g/L Dextrose, 15 g/L Agar, (pH
5.6)] was used to cultivate the fungus. Similarly, Nutrient Agar Medium (NAM)
[5.0 g/L Peptone, 3.0 g/L Beef extract, 5.0 g/L Nacl, 15 g/L Agar, (pH 7.0)]
was prepared for cultivated the bacterium culture. The pH of the medium was
adjusted as standard before autoclaving at 121°C for 15 minutes.
Fermentation
The liquid
medium was prepared without adding agar (PDB or NBM); this prepared medium is
used to cultivate microbes. In this manner, inoculums were prepared by
introducing separately all individual fungal plugs of 1.0 cm in diameter into
250 ml Erlenmeyer flasks containing 100 ml of PDB medium. The inoculated flasks
were incubated in the dark at 25°C in static condition for 15 days.
Extraction
According
to Tiwari et al. (2010) and Yenn et al. (2017), the procedure was carried out
with some minor modifications. After the incubation period, After two weeks of
cultivation, the fermented broth and fungal biomass were separated using
Whatman No.1 filter paper. The cultures of mycelium separated, and the filtrate
was harvested. The collected crude filtrates used potential bioactive
compounds. The obtained extract was dried for the solid plate, and crude extract
paste was obtained. A control was also prepared using a sterile medium
following the same procedure used for fermentative broth.
Antimicrobial Assay:
Disc diffusion assay
The
antimicrobial efficacy of the extract was evaluated according to standards
created by the Clinical Laboratory Standards Institute (CLSI) and Yenn et al.
(2017) with some modifications. The assay was performed by transferring the
fungal inoculums to the surface of nutrient agar medium after 3 to 5 days of
appropriate incubation, inoculated with previously grown bacterial suspension,
allowing all plates to be incubated at 37oC for 24 hrs.;
furthermore, measured the inhibitory zone by antibiotic zone scale. In the
control NAM plate, they have only transferred bacterial suspension. The
experiment is consecutive and was done twice times.
Broth Dilution assay
The
potential inhibitory concentration was determined by using the broth dilution
assay method in different time intervals, according to followed Yenn et al.
(2017) and Chandrawanshi & Shukla (2019). Then, previously fully grown
bacteria containing NBM medium were added to Penicillium mycelium
filtrate extract in equal amounts. These tested tubes were incubated for the
next 24 hrs and absorbed recorded at 600 nm for the following treated six days.
The bacteria containing NBM with an equal amount of mycelium extracts were used
as blank. The minimal lethality concentration was recorded as the lowest
extract concentration to kill the test microorganisms, with dose exposure assayed.
Results
Disc diffusion assay
The
finding of the best suitable Penicillium species was
characterized on a solid medium, and significant antimicrobial activity was
investigated. The present study found that the highest level of antimicrobial
activity was produced by P. oxalicum and P. purpurogenum, as
observed from the results obtained by the agar diffusion method; the zone
diameter has revealed in Table 1. The activity was considered for inhibition
zones wider than 10 mm into the agar surface. The activity against
gram-positive (coccus) soil-isolated bacteria. Followed by P. chrysogenum recorded
only 8 mm diameter, and the minor zone was observed for P. notatum and P.
funiculosum (5 mm); it showed that in the plate assayed, a
minute quantity of penicillin substance secreted, which was seen that
antibiotic inhibitory zone in the plate. The experiment has taken other species
that responded very ineffective, thus considered non detective (nd).
Table 1: Antibiotic sensitivity test, measured of the zone
of inhibition
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SN
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Microorganism
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Diameter (in mm)
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1
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P.oxalicum
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10
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2
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P.notatum
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5
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3
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P.purpurogenum
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10
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4
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P.funiculosum
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5
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5
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P.chrysogenum
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8
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6
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P.notatum
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nd
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7
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P.rubrum
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nd
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8
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P.rugulosum
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nd
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9
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P.citrinum
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nd
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10
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P.variabile
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nd
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nd-Not detected
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Figure 2: Antibacterial activity by Penicillium
filtrates against Gram positive bacteria
Broth Dilution assay
The test
microorganisms exhibited susceptibility to fungal extract from fermented Penicillium culture.
The initially optimized inhibitory activity on a solid medium was further
tested on broth dilution. The assay was performed in a sterile test tube,
according to Yenn et al. (2017). The nutrient broth was used for testing
bacteria, and mycelium filtrate-containing potato broth was used to examine
antibiotic properties. The study showed that P.notatum and P.
citrinum showed very little effectiveness on broth assayed because
optical density observed data showed in increasing order, indicating that the
mycelium filtrate has not potent effective against bacterial culture. P.oxalicum and P.
funiculosum showed the hard capacity of antibacterial properties
against gram-positive bacteria, has observed that the decreasing order and
particular day after the reading was obtained in zero values. It is potent in
both assay system broth and solid surface medium. Then other tested fungi
species, including P. rubrum, P. frequent, P. variabile, P.
rugulosum, P. chrysogenum and P. purpurogenum showed
less effective or insignificant antibiotic properties (figure1), thus not
observed proper growth inhibition activity, similar to the data were expressed
on solid plate medium. Minimal lethality concentration (MLC) was determined
based on the exposure of extract to kill the test microorganisms.
Discussion and Interpretation
The
present study used potato dextrose medium for crude antibacterial compounds
extract successively obtained. The growth media and growth incubation
conditions strongly affect secondary metabolite production, and there is no
compromise on which culture media are optimal for specific metabolite
production. Several culture media like Czapek-Dox broth, Sabourod broth, potato
dextrose broth, malt extract broth, and nutrient broth were used to investigate
previously. However, potato dextrose broth was a potent medium for producing
bioactive antimicrobial metabolites (Mathan et al., 2013). Singh et al. (2012)
and Toghueo et al. (2018) reported the potato dextrose agar is the best medium
for the maximum growth and production of metabolites. Talia et al. (2011)
investigated in vitro synergism between several chalcones
substituted in combination with oxacillin, an antibiotic used conventionally
against S. aureus ATCC 43 300 that was resistant to
meticillin, using the kinetic turbidimetric method developed earlier. The
results were satisfactory for all assayed combinations and followed the
mechanism of bacteriostatic inhibition. Even so, the size of inhibition zones
varied among all microbes, indicating different susceptibility of the test
microorganism to the extract. Among the microbial species, the largest clear
zone was shown by Streptococcus sp., a gram-positive
bacterium. The results obtained supported previous studies that most of the
fungal extracts generally exhibit lower antimicrobial activity against
gram-negative bacteria than gram-positive bacteria (Mahadevamurthy et al., 2016).
Sadrati et al. (2013) reported that the antibacterial activity by plug agar
method for the PLR9 isolate might be due to the secretion of diffusible
extracellular metabolites in the agar medium, these compounds having a specific
effect against bacteria. This technique was easy and widely used to detect
non-volatile compounds produced by microorganisms. The significant
antibacterial activity obtained in the present study may be due to the presence
one or more these molecules in the crude filtrate extracts especially
penicillin acid, which is active against bacteria (Zainuddin et al., 2010;
Varga et al., 2015). Penicillin is secondary metabolites, its production
varying by cultivated factors. Thus, the expression of secondary metabolites
might depend on the culture conditions and the strains. P. corylophilum was
often isolated from temperate climates (Malmstro¨m et al., 2000). However, in
work, they investigated and reported that the strain was isolated from a
Brazilian soil sample, a tropical country. According to Gaden-Junior (2000), metabolite
production is influenced by medium composition, nutrients availability and
others aspects. Microorganisms can use a wide variety of carbon and nitrogen
sources. Neverthe less, many secondary metabolic pathways are negatively
affected by these sources favorable for growth (Cutler et al., 1989; Sanchez
& Demain, 2002). Thus might be the reason for various antibacterial
activity expressions among Penicillium species.
Conflict of Interest
The
authors declare that there is no conflict of interest.
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