Antifungal agent production from a new marine Bacillus pumilusSMH101

Twenty two (22) marine bacterial strains were isolated and tested to inhibit some plant and human pathogenic fungi; Fusarium solani, Rhizoctonia solani, Aspergillus niger, Fusarium exosporium and Candida albicans. The most potent marine bacterium was identified as Bacillus pumilusSMH101 on using 16S rRNA. The peptone water medium (PW) showed maximum antifungal activity. The PlacketBurman experimental design was applied and the optimum culture conditions were inoculum size (8.0 × 10 6 cfu/ml), temperature (25°C), incubation period (24 h) and pH value of 7.0. The trickle flow column was tested for propagating the antifungal production using luffa pulp and synthetic sponge as solid packing materials. The adsorbed B. pumilusSMH101 showed lower average fungal suppression (~ 40%) compared to the free bacterial cells (58.6%). Thin layer chromatography (TLC) was applied using a solvent system of dichloromethane: methanol: water (65:32:3 v/v). A single ultraviolet (UV) spot was obtained with a retardation factor (Rf) of 0.75. It analyzed using UV, infrared (IR) and mass spectrometry (MS) spectra and showed a molecular weight of 875 Da. Also, it showed a relatively low bio-toxicity (LC50 = 1072 ppm) and a broad antifungal spectrum with a bioactivity of 87, 80 and 70% against F. solani, R. solani and C. albicans, respectively, compared to some commercial antifungal drugs based on the active fluconazole compound which applied in a concentration of tenfold more than the used B. pumilusSMH101 antifungal agent concentration (0.05 mg/ml).


INTRODUCTION
In recent years, the incidence of invasive opportunistic fungal infections has been increasing due to increases in the number of immune-compromised patients (Soeta et al., 2009).Infections due to Candida species in immunecompromised patients are the most common; however, Aspergillus and other pathogenic fungi are also emerging as a threat to public health (Richardson, 2005).The mor-tality rate due to invasive aspergillosis has risen stea-dily with a 35.7% increase which caused significant mor-bidity and mortality during 1980 to 1997 (Rapp, 2004).Infecions due to Fusarium species are emerging hyalo-hyphomycoses of immune-compromised patients and are associated with high mortality (Nucci and Anaissie, 2006).The dismal prognosis of Fusarium infection is the result of as, the immobilization technique.A partial purification and characterization of the produced antifungal agent was carried out and the LC 50 of the bioactive spot was determined using Artemia salina as a biomarker.

Isolation and purification of antifungal producer(s)
Seawater samples were collected from different locations in Alexandria shore line using 500 ml sterile blue screw-caped bottles according to the standard methods published by American Public Health Association (APHA, 1995).Serial dilutions were made using filtered sterilized seawater (from 10 -2 to 10 -6 ).A portion (0.1 ml) from each diluted sample was spread on seawater nutrient agar plate medium (5 g peptone, 3 g beef extract, 20 g agar, 1000 ml seawater).Plates were incubated at 30°C for 24 h.A purification of the obtained bacterial colonies was carried out by streaking technique.The purified bacterial colonies were kept separately and tested to inhibit the pathogenic fungi; A. niger, F. solani, P. oxilacum, R. solani and C. albicans ATCC 14053.

Bioactivity test
The bioactivity of each bacterial isolate against the pathogenic fungi was estimated as follows; a suspension of 24 h old bacterial culture (OD ~ 1.0) was centrifuged at 12000 rpm for 10 min.Then 5 ml of each bacterial supernatant was added to 100 ml (CYEA) medium and poured using 9 cm sterile Petri dishes (amended (CYEA) plates).0.5 cm cylinders with 5 mm diameter were cut out from preactivated pathogenic fungi using a sterile cork borer, and then transferred separately to the center of the amended (CYEA) plates and the control (un-amended (CYEA) plates); all plates were incubated at 28°C for a week.The suppression percentage was calculated as follows: (fungal growth diameter on un-amended (CYEA) plates in mm -fungal growth diameter on amended (CYEA) plates in mm)/ fungal growth diameter on un-amended (CYEA) plates in mm × 100% (APHA, 1995).
While, the bioactivity against C. albicans was carried out as follows: 5ml of each bacterial supernatant was added to 100 ml (SDA) medium and poured using 9 cm sterile Petri dishes (amended (SDA) plates).100 µl of C. albicans suspension (OD ~ 1.0) was added to amended (SDA) and the control (un-amended (SDA) plates).
All plates were incubated at 30°C for 48 h.The suppression percentage was calculated as follows: (No. of colonies on unamended (SDA) plates in cfu/ml -No. of colonies on amended agar plates in cfu/ml)/ No of colonies on un-amended (SDA) plates × 100% (APHA, 1995).

Molecular identification process
This process was carried out at National Research Institute, El-Doky, Cairo, Egypt.

DNA extraction
Total DNA content was extracted from overnight pure culture of the most bioactive marine bacterial isolate using Qiagen DNeasy kit (QIAGEN-Inc., Germany) and Genomic DNA purification kit (Promegal).The procedure was identical to that recommended by the manual instructions.The preparations were analyzed on a 0.7% agarose gel and then determined spectrophotometry (Sambrook et al., 1989).

Nucleotide sequence and the accession number
The 16S rRNA gene of a pure culture of the most bioactive marine bacterial isolate generated in this study was sequenced and compared to the database presented at the GenBank.

Optimization of antifungal production using the Plackett-Burman experimental design
Seven independent variables including three medium components; Tryptone, Peptone and NaCl, they were tested in three different concentrations 4, 5 and 6 g/l.In addition, four physiological conditions including; temperatures (25, 30 and 45 o C), pH values (6, 7 and 8), inoculum sizes (8.0 × 10 6 , 7.3 × 10 7 and 9.8 × 10 8 cfu/ml) and incubation periods (24, 48, and 72 h) were also tested.These seven independent variables were screened in nine combinations organized according to the Plackett-Burman design matrix.For each variable, a high (+) and low (-) level was tested.All trials were performed in triplicates and the averages of observed activities were treated as the response.The main effect of each variable was determined using the following equation: Where, Exj is the variable main effect, Mi+ and Mj-are  of suppression percentages of fungal growth in trials where the independent variable (xi) was present in high and low levels, respectively, and N is the number of trials divided by 2. Verification of validity of the optimum medium compared to the basal medium and the Plackett-Burman reverse medium was applied.The t-test statistical analysis was performed for equal unpaired samples to determine the variable significance (Bie et al., 2005).

Application of a trickle flow column bioreactor for antifungal production
In this part of work a trickle flow column bioreactor connected with a peristaltic pump was used to perform antifungal agent production.It was composed of a glass column (60 cm long, 5 cm internal diameter and a total workable volume of 500 ml), it was also connected at the top with an air pump having a flow rate of 1.0 L/min through sterilized air filters.The column was separately packed with 2 to 5 g of the tested solid supporters (sponge and Luffa pulp particles).The packed column and its parts were sterilized using an autoclave for 30 min.Then the sterilized packed column was inoculated with 100 to 150 ml of the antifungal producer suspension (8.0 × 10 6 cfu/ml), and incubated at 25°C till a complete adsorption process of cells was observed.The optical density and pH were continuously detected in the effluent and the efficiency of the system was determined (El-Naggar et al., 2004).

Scanning electron micrograph
Scanning electron micrographs of the used solid supporters (sponge and luffa-pulp) with and without the adsorbed bacterial cells were captured at the Electron Microscopy Center, Faculty of Science; Alexandria University, Egypt.
The obtained spots were separately scratched and dissolved in dimethylsulfooxide.Then Silica gel was removed by centrifugation at 5000 rpm for 15 min and then the antifungal activity of each extract against the tested pathogenic fungi was estimated as mentioned before.

Bio-toxicity of the purified antifungal agent
The toxicity bioassay was carried out according to Meyer et al. (1982) using 24 h old neuplii of Artemia salina as a biomarker.Different concentrations of the purified agent (100, 200, 1000, 1500, 2500 and 5000 ppm) were made and distributed separately in triplicate using clean and dry glass vials (20 ml) then completed to a total volume of 10 ml/each using sterile seawater.Ten live neuplii of A. salina were transferred to each vial.The number of the viable biomarker was counted after 24 h of application.The mortality percentages and the half lethal concentration (LC50) were determined using the probit analysis method (Reish et al., 1987).

A Comparison between the antifungal activity of the purified agent and some commercial antifungal drugs
The bioactivity of 50 ppm the purified antifungal component in comparison to the bioactivity of 500 ppm/each of three commercial antifungal drugs based on the active fluconazole compound (Diflucan, Flocoral and Fungimycin) was estimated using A. niger, F. solani, P. oxilacum, R. solani and C. albicans.The suppression growth % of the pathogenic fungi was detected compared to the control (untreated fungi).

Partial chemical characterization of the purified antifungal agent
The UV/Visible spectrum of the most active spot was determined using spectrophotometer UV-vis./Jenway6800 and the dimethylsulfooxide solvent as a blank.The infrared spectrum of the antifungal agent was carried out using a Peak Find-Memory-27 spectrophotometer at the Microanalysis Center, Cairo University, Egypt.The Mass spectrum of the antifungal agent was subjected using DI Analysis Shimadzu Qp-2010plus/mass spectrophotometer at the Microanalysis Center, Cairo University, Egypt.

Isolation and molecular identification of the most potent marine antifungal producer
A preliminary analysis for antifungal activity of different marine bacterial strains isolated from the eastern harbor and the western harbor of Alexandria, Egypt was conducted using some plant and human pathogenic fungi.All strains were grown on nutrient broth medium and then screened to select the most potent marine bacterial isolate acting against at least three pathogenic fungi with an activity >30%.It was showed that the marine bacterial isolate coded MS12 was the most potent isolate acting against the tested plant and human pathogenic fungi, F. solani, R. solani and C. albicans, the antifungal activities were 50, 43 and 32%, respectively.
The obtained amplified PCR fragment (835 bp) was purified and detected using agarose gel electrophoresis.
Then the obtained amplified 16S rRNA was sequenced and compared with the data presented in the Genbank using Blast search program.It was found that the bacterial isolate MS12 had a new genomic sequence which indicate the isolation of a new strain of Bacillus pumilus, it genetically identified as Bacillus pumilusSMH101 with a new association No.KF964031.

Effect of different culture media
Five different culture media were examined to obtain the highest antifungal activity from B. pumilusSMH101.It was found that the peptone water medium was the more effective medium tested compared to others.The percentage of the antifungal activity against F. solani, R. solani and C. albicans was 52.5, 47.1 and 34.9, respectively, (Table 1).

Application of Plackett-Burman design
The components of B. pumilusSMH101 culture medium in addition to the physiological conditions were optimized for a maximum antifungal activity against F. solani, R. solani and C. albicans; it was carried out using the Plackett-Burman experimental design (Table 2).
The main effect of the tested variable was presented as the difference between the fungal suppression % averages at both the high level (+) and the low level (-) of the examined variable (Figure 1).The obtained data of the main effect as well as the t-test values showed the physiological conditions; inoculum size, temperature and the incubation period must be adjusted at their low levels (8.0 × 10 6 cfu/ml, 25°C and 24 h, respectively) to obtain more antifungal activities against F. solani, R. solani and C. albicans.Moreover, the inhibition of both F. solani and R. solani was maximized on the addition of the high level (6 g/L) of tryptone, while, the inhibition of C. albicans was maximized on adding the high level (6 g/L) of both peptone and NaCl.

Verification of Plackett-Burman experiment
In order to validate the obtained results and to evaluate the accuracy of the applied Plackett-Burman statistical design, a verification experiment was carried out in triplicates to predict the near optimum levels of independent variables.The data were examined and compared to the basal and anti-optimized medium.It was revealed that the average antifungal activity against F. solani, R. solani and C. albicans by B. pumilusSMH101 was increased by 1.5, 1.5 and 1.9 fold, respectively, when grow on the optimized medium (data not shown).The results indicated for inhibiting both F. solani and R. solani the  culture should be formulated as follows (g/L): tryptone (6), peptone (5), NaCl (5), in addition, pH (7.0), inoculum size (8.0 × 10 6 cfu/ml), temperature (25°C) and incubation period of 24 h.While, for inhibiting C. albicans the formula should be as follows: (g/l): tryptone (5), peptone (6), NaCl (6), in addition, pH (8.0), inoculum size (8.0 × 10 6 cfu/ml), temperature (25°C) and incubation period of 24 h.

Application of trickle flow column
A trickle flow column bioreactor connected with a peristaltic pump was used to perform antifungal agent production.The column was separately packed of the tested solid supporters (sponge and luffa pulp particles).At the end of the incubation period, the antifungal activity was determined and compared to free cells of B. pumilusSMH101.The adsorbed cells on sponge particles led to antifungal activities of 50.9, 45.4 and 29.2% against F. solani, R. solani and C. albicans, respectively.The adsorbed cells on luffa pulp particles led to antifungal activities of 47.2, 42.2 and 23.8%, respectively, (Table 3).Moreover, the development of the bacterial biofilms on these used solid supporters was investigated using scanning electron microscopy (Figure 2).

Bio-toxicity of the B. pumilusSMH101 purified extract using A. salina as a biomarker
The bio-toxicity of different concentrations (from 100 to 5000 ppm) of B. pumilusSMH101 purified extract were estimated using A. salina as a biomarker, then the mortality percent was calculated and presented using the probit analysis method.The results indicated the purified antifungal agent had a relatively low toxicity level (Table 4).The LC 50 (the concentration at which 50% of the tested biomarker individuals die) of this agent was 1072 ppm and it was estimated from the best fit line obtained on using the probit analysis method.

A comparison of the antifungal activity of the partially purified agent of B. pumilusSMH101 to some commercial antifungal drugs
The bioactivity of 50 ppm purified antifungal agent of the marine B. pumilusSMH101 was separately compared to 500 ppm of each commercial antifungal drug; this concentration is tenfold more than that of the produced antifungal agent of the marine B. pumilusSMH101.The data presented in Table 5 showed a broad antifungal spectrum of the purified agent of B. pumilusSMH101 with an average activity of 60% against the five tested human and plant pathogenic fungi compared to the tested commercial drugs based on the active fluconazole compound; Diflucan, Flocoral and Fungican; the obtained average inhibition percentage was 48, 49 and 54%, respectively.Moreover, the most inhibited fungus was F. solani (87%) followed by R. solani (80%) and C. albicans (70%) compared to the tested antifungal drugs; the average inhibition percent was 60, 55 and 70%, respectively.Moreover, the photographs in Figure (3) showed the inhibition percent obtained by the purified marine B. pumilusSMH101 agent (Figure 3-B) compared to the untreated fungi (control) (Figure 3-A).

Partial Characterization of the B. pumilusSMH101 purified antifungal agent
The chemical characterization presented in Figure ( 4) showed the UV-Vis, infra-red (IR), and Mass spectra of the purified antifungal agent.The UV spectrum of the compound (Figure 4-A) resulted in a single peak appeared at ƛ 280 nm which proved the aromatic character of the compound.The IR spectrum showed seven absorption bands (Figure 4-B) the major five bands were  explained as follows; the first band appeared at 3441 cm which indicated the presence of NH 2 , OH or NH groups, the second band appeared at 2966.95 cm which indicated the presence of the aromatic C-H group, the third a The value of LC50 was detected using the probit analysis, it was 1072 ppm.
third band appeared at 1638.23 cm which indicated the at 1457.92 cm which indicated the presence of aromatic ring in the compound.The last band appeared at 1051.01 cm which indicated the presence of the ether linkage in this compound.Moreover, the obtained Mass spectrum of this compound (Figure 4-C) showed the appearance of a molecular ion peak at m/e = 888.40,while, the base ion peak was appeared at m/e =132.10.The molecular weight of this compound showed to be 875Da.

DISCUSSION
Genus Bacillus has a long and distinguished history in the field of biotechnology.Since, members of this genus are used for the synthesis of a very wide range of important medical, agriculture, pharmaceutical and other industrial products including antibiotics, bacteriocins, enzymes, amino acids, sugars, surfactants and flavor enhancers (Anthony et al., 2009;Bhaskar et al., 2007;Lisboa et al., 2006;Parvathi et al., 2009;Ying et al., 2005).
Table 5.The antifungal activity (%) of the partially purified agent of B. pumilusSMH101 (0.05 mg/ml) compared to the antifungal activity (%) of some commercial antifungal drugs based on the active fluconazole compound (0.5 mg/ml) using different plant and human pathogenic fungi.Statistical experimental designs are powerful tools for an economic and a rapid search of the key factors from a multivariable system.It minimizes the error in determining the effect of these variables on the growth of the used microorganisms (Abou-Elela et al., 2009;Xiong et al., 2007).So, Plackett-Burman experimental design was used to optimize the components of the culture medium and reflect the relative importance of various environmental factors on the production of the antifungal agent by B. pumilusSMH101 in liquid cultures.It was found the optimum physiological conditions for antifungal agent production by B. pumilusSMH101 were achieved on applying the tested low levels; the temperature was 25°C, the inoculum size was 8.0 × 10 6 cfu/ml and the incubation period was 24 h, regardless the target pathogen.While the optimum culture components showed insignificant effect on the production of the antifungal agent by B. pumilusSMH101 and they varied according to the pathogenic target (Figure 1 and Table 2).

Types of antifungal
Cell immobilization shows many operational and economic advantages such as prolong metabolic activities, reuse of the biocatalyst, increase of cell concentration in preventing washing out of cells at high flow rates (Gautam et al., 2002).However, the results obtained on using immobilized B. pumilusSMH101 cells (absorbed bacterial cells on solid supporters; luffa pulp and sponge) showed low antifungal activities against F. solani, R. solani and C. albicans.The average activity of the adsorbed cells was 37.8 and 41.8%, respectively, compared with the free bacterial cells of B. pumilusSMH101 58.6% (Table 3).Similar results were obtained by Freeman and Aharonowitz (1981) who developed a mild new method for the immobilization of the whole microbial cells.They found the yield of cepha losporins antibiotic production by the immobilized bac-terial cells using acrylamide monomers (direct poly-merization method) was significantly decreased com-pared to the free cells of Streptomyces clavuligerus.Moreover, Punita et al. (2007) used Immobilized Acremonium chrysogenum (mold) cells for cephalo-sporin-C production.It was found that cell growth rate of immobilized cells was reduced with about 39% of the growth rate of free cells.Contrary, Srinivasulu et al. (2003) studied the immobilization effect of Streptomyces marinensisNUV-5 using calcium alginate for the production of neomycin.They reported the antibiotic productivity was enhanced with 32% on the use of the immobilized cells over the use of the conventional freecell.
The fungicidal activity of another B. pumilus strain was investigated by Bottone and Peluso (2003).They found that a compound produced by B. pumilus inhibits Mucor and Aspergillus species through the inhibition of spore germination and aborted elongating hyphae.The molecular mass of this compound was determined by diffusion through dialysis membrane to be 500 to 3000 Da.These findings were agreed with the results obtained in this study; the photographs of the purified antifungal agent indicated the fungicidal action against F. solani and R. solani was carried out through inhibiting the hyphae elongation (Figure 3), the bioactivity was 87 and 80%, respectively, compared to the control (Table 5).Also, the determined molecular weight (875 Da) of this antifungal agent through MS-spectrometry was located between 500 and 3000 Da.Also, Aunpad and Na-Bangchang (2007) isolated B. pumilusWAPB4, it showed a remarkable antibacterial activity against methicillin-resistant Staphylococcus aureusMRSA, vancomycin-resistant Enterococcus faecalisVRE, and several Gram-positive bacteria.This Bacteriocin was designated as pumilicin-4 with a molecular mass of 1994.62Da using a mass spectrometry.
Moreover, the obtained B. pumilusSMH101 antifungal agent found to have an antagonistic action against the tested C. albicans with a bioactivity of 70% compared to the control (Figure 3).Contrarily, Guo et al. (2009) used thymol (THY) which was found to have in vitro antifungal activity against 24 fluconazole (FLC)-resistant and 12 FLC-susceptible clinical isolates of C. albicans but no antagonistic action was observed.
In general, the results of the bio-toxicity test of the obtained purified antifungal agent of B. pumilusSMH101 showed a relatively low toxicity level where the LC 50 was 1072 ppm which is much more than the concentration used in the application process of this study (Table 4).Dissimilarly, many authors worked on using B. pumilus in order to obtain bioactive secondary metabolites acting against different pathogenic fungi regardless the biotoxicity of these metabolites towards human or plant (Ghasemi et al., 2012;Munimbazi andBullerman, 1997, 1998;Munimbazi and Bullerman, 1998).On the other hand, Yadav et al. (2007) purified a cytosolic protein from E. coli BL21; it demonstrated potent antifungal activity against pathogenic strains of Aspergillus species (A.fumigatus, A. flavus, A. niger) and C. albicans with MIC of 1.95 to 3.98 and 15.62 mg ml -1 , respectively, and it showed no cytotoxicity up to 1250 mg ml -1 in vitro toxicity tests, which is very high concentration compared to the LC 50 of the antifungal agent of B. pumilusSMH101 (Table 4).
Finally, from the results of the comparative study it can concluded this produced safe antifungal agent from the  marine B. pumilusSMH101 may act as a promising alternative tool for the treatment of some pathogenic fungi; F. solani, R. solani and C. albicans even on using a very low concentration (0.05 mg/ml) (Table 5), this is to face the increasing in the pathogenic fungus resistance towards the used commercial antifungal drugs especially those based on the active fluconazole compound.On the other hand, many investigations will be carried out in order to precisely identify the main active component in this produced antifungal agent.

Figure 2 .
Figure 2. Micrographs show the development of the adsorbed B. pumilusSMH101 biofilm on sponge (S) and luffa pulp (L) as packing supporters of the used glass trickle flow column after 2 days (B), 4 days (C), and 6 days (D) compared to the uninoculated supporters (control) (A).

Figure 3 .
Figure 3. Photographs show the antifungal activities of the purified marine B. pumilusSMH101 agent (B) against C. albicans (Ca), R. solani (R) and F. solani (F) compared to the untreated fungi (control) (A).

Figure 4 .
Figure 4. Some chemical characterization of the antifungal active component produced by B. pumilusSMH101 using dimethylsulfooxide (DMSO) as a blank.UV/Vis.Spectrum (A), IR spectrum (B) and Ms Spectrum (C).

Table 1 .
The effect of different culture media on the antifungal activity (%) of B. pumilusMSH101 using some plant and human pathogenic fungi as indicators.

Table 2 .
The optimization of the antifungal production by B. pumilusSMH101 using the Plackett-Burman experimental design and its antifungal activities against some plant and human pathogenic fungi.

Table 3 .
Antifungal activity (%) of the adsorbed B. pumilusSMH101 cells against F. solani, R. solani and C. albicans using a glass trickle flow column and the optimized culture medium.

Table 4 .
The bio-toxicity of different concentrations of the purified antifungal agent of B. pumilusSMH101 using Artemia salina as a biomarker.