Antibacterial activity of endophytic fungus , Penicillium griseofulvum MPR 1 isolated from medicinal plant , Mentha pulegium

In the present study, an endophytic fungus with a considerable antimicrobial activity was isolated from the medicinal plant Mentha Pulegium L. The morphological and molecular identification revealed that this fungus is a Penicillium griseofulvum. A preliminary screening was done to choose the suitable culture medium for a maximum production of the bioactive compounds using the dual-culture agar diffusion assay. The malt extract agar (MEA) and potato dextrose agar (PDA) media were the suitable, and the MEA was selected for the further study. Extraction was done with three solvents, n-Hexane, dichloromethane and ethyl acetate, and the crude extracts were tested against Gram-positive and negative bacteria. A high activity was found with ethyl acetate and dichloromethane crude extracts against all bacteria with a maximal inhibition zones of 45.5 and 41 mm respectively obtained against Escherichia coli. The minimum inhibitory and bactericidal concentrations (MIC, MBC) of ethyl acetate crude extract were evaluated using the broth micro-dilution method. A MIC of 50 μg / ml on Gramnegative bacteria and of 100 μg / ml on Gram-positive bacteria was found. The MBCS ranged from 50 and 200 μg/ml. The time kill study has revealed a bactericidal activity of the Penicillium griseofulvum crude extract. At 24 h and for all concentrations (MIC× 2 and MIC× 4), 100% killing of the bacterial cells was achieved. These results prove that the extract of Penicillium griseofulvum can be a promising source of important bioactive molecules.

. Endophytic fungi live asymptomatically in the intercellular and/or intracellular spaces of healthy plants for at least a part of their life (Dos Banhos et al., 2014).All parts (leaves, stems, roots, barks, etc) of all plants examined till date are colonized by endophytic fungi (Shen et al., 2014).The relationships between the endophytic fungi and their hosts can change between neutral association to mutualistic, pathogenic or saprophytic interactions depending on genetic dispositions of the two partners, environmental factors and time (Martín et al., 2013).
The increasing resistance of pathogenic microorganisms to antibiotics in the world, and recently in Algeria, leading to the resurgence of more complex infections (Baba Ahmed-Kazi Tani and Arlet, 2014), has pushed researchers to discover new bioactive molecules.The capacity of endophytic fungi to biosynthesize bioactive secondary metabolites (more than 20.000 bioactive molecules) (Marson Ascêncio et al., 2014) and their various biological activities such as antimicrobial, anti-viral, anticancer, antioxidant, antidiabetic, and antiinflammatory activity (Sadrati et al., 2013;Bashyal et al., 2014;Shah et al., 2015;Jouda et al., 2016a;Pan et al., 2016;Chen et al., 2016;Singh and Kaur 2016;Yao et al., 2017) have made this fungus to be important, as an important source of new bioactive secondary metabolites potentially useful for human medicine and other domains.
Medicinal plants have been well studied because they harbor rare microorganisms, that imitate the chemistry of their hosts, and making the same molecules or derivate more active than those synthesized by their hosts.It is also probable that the bioactivity of medicinal plants may be due to endophytes living inside plants by genetic exchange (Tayung et al., 2011).
Mentha pulegium L., commonly known as pennyroyal, belongs to the Lamiaceae family.It is native to America, Europe, North Africa and in Asia Minor and Near East (Mahboubi and Haghi, 2008).It is widely used in traditional medicine (food poisoning, bronchitis, diuretic, flatulence, intestinal colic), food like culinary herb, cosmetics and aromatherapy (Hajlaoui et al., 2009;Aires et al., 2016).Only Debbab et al. (2009) and Teiten et al. (2013) have studied the endophytic fungi of this plant, and have determined the cytotoxicity and anticancer activity of the bioactive metabolites of the endophytic fungus, Stemphylium globuliferum.However, the antibacterial activity of endophytic fungus of this plant has not been examined; for this reason we selected this plant for isolation of this endophytic fungus in order to investigate its antibacterial activity.

Sample collection and fungal isolation
Endophytic fungi were isolated from the healthy medicinal plant Mentha pulegium L., collected from the mountain of Megriss Setif Zerroug et al. 1057 Algeria in June 2014.They were identified by faculty botanists, according the following method.Different tissues (leaves, stems and roots) were washed with tap water, cut into small pieces (leaf pieces size of approximately 5.0 × 5.0 cm; stems and root pieces of approximately 5.0 cm long); they were surface-sterilized by sequential immersions in 70% (v/v) ethanol for 1 min, 3% sodium hypochlorite for 4 min and 70% (v/v) ethanol for 1 min and then rinsed thrice in sterilized distilled water for 1 min each (Petrini, 1986;Pimentel et al., 2006;Xie et al., 2016).After drying on sterile filter paper, each segment was cut into 0.5 cm and aseptically transferred onto Petri-dishes containing Potato Dextrose Agar (PDA) (Sigma-Aldrich; pH 5.6 ± 0.2) supplemented with 100 mg/l of Gentamicine to suppress bacterial growth.The dishes were incubated at 28°C and checked continuously for the growth of endophytic fungi colonies.Each fungus emerging from the segments was transferred to fresh PDA plates and PDA slants for conservation at 4°C.

Preliminary antibacterial assay
Antibacterial activity of the endophytic fungi was performed using the method of agar plug diffusion explained by Powthong et al. (2013) and Sahani et al. (2017), with some modification.Briefly, pure cultures of the endophytic fungi were cultivated on the surface of PDA at 28°C for 14 days.Then, a small disk of fungal colony was cut (6 mm diameter) using a sterile cork borer and placed on previously inoculated Muller Hinton Agar (MHA) (Sigma-Aldrich; 7.3±0.2) plates.A disk of PDA without fungi was used as negative control.The Petri dishes were refrigerated at 4°C for 4 h for complete diffusion of antibacterial compounds from the fungal disks, then these plates were incubated at 37°C for 24 h.Antibacterial activity was determined by measuring zones of inhibition produced by the endophytic fungi.The endophytic fungus MPR1 displaying good antibacterial activity was selected for further study.

Identification
The fungus was initially identified based on morphological characteristics using the standard mycological manuals and microscopic examination (Rajeswari et al., 2017).For molecular identification, isolate was grown on PDA plate for 7 days at 25°C.DNA was extracted from culture using the Ultraclean Microbial DNA Isolation Kit (MoBio, Solana Beach, CA, USA) following the manufacturer's protocols.Molecular characterization of the isolates was performed using sequencing of the internal transcribed spacer (ITS) region that is standard gene regions.For this aim, ITS gene regions were amplified with the primers V9G, 5'-TTACGTCCCTGCCCTTTGTA-3' (forward) and LS266, 5'-GCATTCCCAAACAACTCGACTC-3' (revers) (Chen et al., 2016;Kadaifciler and Demirel, 2017) by polymerase chain reaction ].

Data analyses
The sequence was compared with those deposited in the NCBI GenBank Database via BLAST searches (Altşchul et al., 1990), and aligned using the Muscle in MEGA7.0 software package together with the other sequences of morphologically and phylogenetically related type species that were obtained from NCBI GenBank (Kumar et al., 2016).The aligned data sets were analyzed using Maximum Likelihood analysis based on the Tamura Nei model (Tamura and Nei, 1993) as implemented in the MEGA 7.0 with 1000 bootstrap replications.Gaps and missing data in all the position were eliminated.Penicillium crustosum (AF033472) was used as out group.The obtained sequence data were deposited in NCBI GenBank, and an accession number was obtained.
In order to choose the best extraction solvent, three organic solvents were used as n-Hexane, dichloromethane and ethyl acetate.The extraction method was performed based on the method explained by Saraswaty et al. (2013).Briefly, the supernatant of liquid fermentation was extracted with an equal volume of organic solvent starting with the non-polar organic solvent (n-Hexane).The solution was then mixed for 10 min and allowed to stand.The layer of n-Hexane was then collected and evaporated under vacuum using a rotary evaporator (BÜCHI).The remaining aqueous phase was re-extracted by the other solvents using the same method.The crude extracts were then dissolved in dimethyl sulphoxide (DMSO) and kept at 4°C.

Evaluation of antibacterial activity of crude extracts
Antibacterial activity of secondary metabolites extracted from MPR1 was done following an agar well diffusion method described by Fatima et al. (2016)  100 µl (10 8 c.f.u./ ml) of bacterial inoculums was spread on MHA plates; then wells (6 mm diameter) were made and 25 µl of each extract was poured into these wells.DMSO was used as negative control.After incubation of 24 h at 37°C, the diameter of each zone of clearance on plates was measured and used as an indicator of antibacterial activity of the extract.
After 24 h of incubation at 37°C, 20 µl of 0.5% of 2, 3-5 Triphenyltetrazolium chloride aqueous solution was added to each well.After a second incubation for 30 min at 37°C, the lowest concentration of ethyl acetate crude extract which did not show any visual color change was recorded as the MIC.All wells that showed growth inhibition were subcultured on MHA.After incubation of 24 h at 37°C, the lowest concentration that exhibited no visible growth was considered to be the MBC.

Time kill study
To determine the bactericidal or bacteriostatic effect of Penicillium griseofulvum ethyl acetate extract, the time kill test was done against Methicillin-resistant S. aureus ATCC 43300.Into three tubes of MHB medium with different concentrations of extract (MIC, MIC× 2 and MIC× 4), the bacterial suspension was transferred to obtain an initial inoculum cell density of approx.5 × 10 5 c.f.u./ ml; a fourth tube was used as the growth control containing the MHB and the bacterial suspension.These four tubes were then incubated at 37°C in a rotary shaker at 150 rev / min for varied time intervals (0, 1, 2, 4, 6, 12 and 24 h).For viable cells counts, in each time interval, a 0.1 ml sample was removed, diluted and spread onto  MHA plates and these plates were then incubated at 37°C for 24 h and the bacterial colonies were counted.Time kill curve (log c.f.u./ ml vs. time) was drawn for each concentration of extract and control culture, and percentages of dead cells were calculated as the following equation: Where, V0 is the initial viable cell count and Vz is the viable cell count at time z (Ibrahim et al., 2015).
Generally, the bactericidal effect is obtained with a lethality percentage of 90% for 6 h, which is equivalent to 99.99% of lethality for 24 h (Balouiri et al., 2016).

Statistical analysis
All experiments were performed in duplicates, and statistical analysis was carried out using SAS/STAT® 9.2 software.Group comparisons were performed using the two-way ANOVA followed by Student-Newman-Keuls multip-rang test.Results are represented as mean ± standard deviation (SD) and significant effects of treatments were determined by F values (P≤ 0.05).

Isolation and preliminary antibacterial assay of endophytic fungi
The 16 endophytic fungi emerging from the surface sterilized plant segments were preliminary screened against four clinical pathogenic bacteria; this permitted a rapid and qualitative selection of the bioactive microorganism.
According to the results of this screening, we focused our work on a single fungal isolate MPR1 that inhibited all of these pathogenic bacteria with a good activity (Table 1).
The isolate showed maximum activity against E. coli ATCC 25922 with an inhibition diameter zone of 47.5 mm followed by B. cereus ATCC 10876, S. aureus ATCC 25923 and P. aeruginosa ATCC 27853 with inhibition diameters zones of 33.5; 31.5 and 16.5 mm, respectively.
The results of this preliminary screening are in agreement with the results obtained with endophytic fungi isolated from two medicinal plants Ocimum sanctum and Aloe vera by Sahani et al. (2017), where all isolated endophytic fungi had broad-spectrum of antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria.

Identification of endophytic fungus MPR1
The fungal isolate MPR1 was characterized by observing colony morphology, pigmentation, growth pattern and sporulation structure, and the fungus has been preliminarily identified up to genus level as Penicillium sp.(Figure 1).
Best-scoring maximum likelihood tree based on the Tamura-Nei model calculated using MEGA 7.0 based on ITS sequences showing the relationships of the newly generated sequences (GenBank accession number MH006592) in this study with previously known taxa in the NCBI GenBank.The scale bar denotes 0.002 substitutions per position.The tree with the highest log likelihood (-1444.9347) is shown.The percentage of trees in which the associated taxa clustered together is shown next to the branches.Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value.The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.The analysis involved 58 nucleotide sequences.There were a total of 451 positions in the final dataset.Figure 2 shows phylogenetic   tree belong to investigated taxa.The endophytic fungus MPR1 was found to be a homolog of type culture Penicillium griseofulvum NRRL 2300 (AF033468).
The tree is rooted with Penicillium lanosum NRRL 2009 (DQ304540) (Bootstrap 1000).The species P. griseofulvum has been isolated as endophyte from different host plants in different studies.For example, Conti et al. (2012) isolated P. griseofulvum from the leaves of S. verticillata (L.).Another study resulted in the isolation of P. griseofulvum from Palicourea Tetraphylla Cham.and Schltdl (Rosa et al., 2010).D 'Souza and Hiremath (2015) isolated P. griseofulvum from seven different medicinal plants of India.

Selection of suitable culture media
For the optimization of the production of the endophytic fungi bioactive molecules of five different basal media PDA, SDA, YEA, MEA, YMEA were used (Table 2).
According to the statistical analysis (Figure 3), MEA and PDA (no significant differences) were found to be the best media for maximum production of antibacterial bioactive compound with average inhibition zones of 33.8 mm and 31.8 mm, respectively and the best activity was observed against Methiciline-resistant S. aureus ATCC 43300 (42.7 mm and 40.3 mm respectively).YEA comes in second position (20.2 mm of average inhibition zones)   with maximal inhibition zone of 27.0 mm obtained against C. freundii ATCC 8090, followed by SDA and YMEA (no significant differences), with average inhibition zones of 15.4, 15.3 and 28.3 mm against S. typhimurium ATCC 13311, and 25.0 mm against E. coli ATCC 25922, respectively.Data were presented as mean ± SD (n=2), means with the same letter are not significantly different (p > 0.05).
Anwar and Iqbal (2017) also studied the effect of growth media on antibacterial activity, and suggested that secondary metabolites are highly affected by the composition of the growth medium.Verma et al. (2017) found that starch was the source of carbon allowing the endophytic fungus to produce the highest amount of bioactive molecules followed by glucose.This is in agreement with the results obtained with MPR1 on PDA and MEA that contain the appropriate concentration of starch and glucose.The same results have been obtained with Mathan et al. (2013), where the PDA was the medium giving the best activity for Aspergillus terreus.

Evaluation of antibacterial activity of crude extracts
Table 3 and Figure 4 show the antibacterial activity of different crude extracts of P. griseofulvum on agar well diffusion method.All the pathogenic bacteria were inhibited by ethyl acetate and dichloromethane crude extracts with the maximal activity showed against E. coli ATCC 25922 (45.5 and 41.0 mm respectively), unlike n-Hexane crude extract which did not inhibit any pathogenic bacteria.
The statistical analysis (Figure 5) showed that ethyl acetate was the best extraction solvent allowing the extraction of bioactive molecules (41.9 mm of average inhibition zones), followed by dichloromethane (35.7 mm of average inhibition zones).These can be explained that the bioactive molecules produced by this fungus are better extracted by polar solvents ethyl acetate and also dichloromethane than apolar like n-Hexane.
Data were presented as mean ± SD (n=2), means with the same letter are not significantly different (p > 0.05).
These results correspond to those obtained by Tong et al. (2014) where antimicrobial compounds of Phomopsis sp.ED2 and the endophytic fungus of medicinal herb Orthosiphon stamineus were mainly present in the ethyl acetate extract.Another study showed that the ethyl acetate extracts of two endophytic fungi isolated from Ocimum citriodorum Vis.show better activity than the other extracts (Mu'azzam et al., 2015).

Determination of minimum inhibitory concentration (MIC) and minimal bactericidal concentrations (MBC)
MIC and MBC values (Table 4) were recorded for the crude ethyl acetate extract from P. griseofulvum; the MIC of this crude extract was of 50 µg / ml for the three Gramnegative pathogenic bacteria (C.freundii ATCC 8090, S. typhimurium ATCC 13311, E. coli ATCC 25922) and of 100 µg / ml for the three Gram-positive pathogenic bacteria (B.cereus ATCC 10876, S. aureus ATCC 25923, Methiciline-resistant S. aureus ATCC 43300).
According to Klepser et al. (1998), if the MBC/MIC ratio is ≤ 4, the extract is bactericidal and if > 4, it is bacteriostatic; and since MBC/MIC ratios obtained for all the test bacteria were ≤ 4 we can say that P. griseofulvum crude extract has a bactericidal effect against all pathogenic bacteria.
Broad spectrum antibacterial activity obtained by this endophytic fungus extract has already been observed with extracts of different endophytic fungi such as the endophytic fungi isolated from Calotropis procera (Rani et al., 2017).Gram-negative bacteria are generally more resistant than Gram-positive bacteria and are therefore more difficult to contend, because their outer membrane protects the peptidoglycan cell wall.The results obtained in our study reveal the opposite, and this can be explained by the fact that the mode of action of the active molecules produced by P. griseofulvum MPR1 is not on the cell wall but possesses a different mode of action (Yenn et al., 2014).These results correspond to the results obtained with the extracts of other Penicillium such as Penicillium sp., an endophytic fungus of Garcinia nobilis that has been a source of new molecules with antibacterial activity mainly on Gram-negative bacteria (Jouda et al., 2014(Jouda et al., , 2016b)).Penicillium minioluteum ED 24 isolated from Orthosiphon stamineus Benth.also show better activity against Gram-negative compared to Gram-positive bacteria (Yenn et al., 2014).

Time kill
The time-kill dynamic process is used for the evaluation of bactericidal or bacteriostatic effect of bioactive molecules; bactericidal activity is indicated by a 3-log 10 reduction, the equivalent of 99.9% cell death, and the number of viable cells in the presence of the antibacterial compound (Balouiri et al., 2016).
Killing growth profile of P. griseofulvum ethyl acetate crude extract against Methicilline-resistant S. aureus ATCC 43300 shows a bactericidal effect at 24 h of incubation; 100% killing of the bacterial cells was achieved for MIC × 2 and MIC × 4 concentrations (Figure 6).

Conclusion
In this study, we highlighted the antibacterial potency of endophytic fungi isolated from the medicinal plant, M. pulegium L. With preliminary screening we selected one isolate P. griseofulvum with a good antibacterial activity.This was confirmed after extraction, where ethyl acetate was the best solvent used for the extraction of bioactive secondary metabolites.The MIC/MBC and time kill of this extract show that it has a bactericidal effect against several bacteria.These promising results push us to want to determine the composition of this extract as well as its active fraction.

Figure 2 .
Figure 2. Best-scoring Maximum likelihood tree calculated using MEGA 7.0 based on ITS sequences showing the relationships of the newly generated sequence in this study with previously known taxa in NCBI GenBank.

Figure 3 .
Figure 3.Effect of different culture media on antibacterial activity of Penicillium griseofulvum against all pathogenic bacteria.

Figure 5 .
Figure 5.Effect of different extraction solvents on antibacterial activity of Penicillium griseofulvum against all pathogenic bacteria.