Prevalence of lipase producer Aspergillusniger in nuts and anti-biofilm efficacy of its crude lipase against some human pathogenic bacteria

Nuts are the natural source of healthy lipids, proteins, and omega-3. They are susceptible to fungal and mycotoxins contamination because of their high nutritional value. Twenty-five species comprising 12 genera were isolated from 80 samples of dried fruits and nuts using the dilution plate method. Peanut recorded the highest level of contamination followed by coconut; almond and raisin were the lowest. Aspergillus was the most prevalent genus and A. niger, was the most dominant species. The morphological identification of the selected A. niger isolates as they were detected in high frequency of occurrence was confirmed by using 18SrRNA sequence. Ochratoxin biosynthesis gene Aopks was detected in the tested isolates. Lipase production by the selected A. niger isolates was determined with enzyme activity index (EAI) ranging from 2.02 to 3.28. A. niger-26 was the highest lipase producer with enzyme activity of 0.6 ± 0.1 U/ml by the trimetric method. Lip2 gene was also detected in the tested isolates. Finally, the antibacterial and antibiofilm efficiency of crude lipase against some human pathogens was monitored. Results exhibited great antibacterial efficacy with minimum bactericidal concentration (MBC) of 20 to 40 µl/100 µl against Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Methicillin-resistant Staphylococcus aureus (MRSA). Interestingly, significant anti-biofilm efficacy with inhibition percentages of 95.3, 74.9, 77.1 and 93.6% was observed against the tested pathogens, respectively.

. Colony forming units (CFU/g), percentage (%C), frequency (F%) and number of cases of isolation (NCI) of mycobiota contaminating nuts. CFU/g: Colony forming unit per gm of 80 samples of peanut, almond, coconut and raisin (20 of each) on rose Bengal chloramphenicol agar medium (RBCA). %C: Percentage of each isolate to the total isolates for each type of nuts. F%: Frequency of each isolate. NCI: number of cases of isolation of each isolate out of 20 sample of peanut, almond, coconut and raisin.
Paecilomyces variotii 0.12 0.6%    (Fig. 6a,b). Other cells were curved and divided like Proteus mirabilis (Fig. 6c,d). Cells of Pseudomonas aeruginosa (Fig. 6e,f) distortion occur in cell shape to spherical instead of bacillus. Finally, cells of Methicillin-resistant Staphylococcus aureus (MRSA) that begin to swell up with irregular spherical shape Fig. 6g,h. SEM micrographs for biofilm structure revealed that, in control, there are typically heterogeneous distributions of biofilm with higher number of adhered cells also cells arranged in the form of aggregates or simply as individualized cells without slimy material in their vicinity (Fig. 7a,c,e,g). In contrast, to treatment with crude lipase where micrographs showing a uniform layer of cells with negligible clumping (Fig. 7b,d,f,h).

Discussion
Nuts and dried fruits are healthful foods that protect human body from many chronic diseases. Their high nutritional value makes them a suitable medium for fungal contamination. In the current study, peanut were the highest contaminated samples this may be due to the high moisture content of peanut samples in harmony with Ismail 28 , who reported that peanut samples were highly deteriorated with fungi than coconut. Aspergillus was the most prevalent genus followed by Penicillium. This were previously confirmed by Khosravi et al. 29 , who showed that Aspergillus followed by Penicillium were the most frequent genera deteriorated 60 samples of nuts. From the above genera A. niger, A. flavus, P. chrysogenum and P. oxalicum were the most frequent species (Table 1) and these results were previously obtained by Ismail 28,[30][31][32][33] . In contrast, Aspergillus section Flavi was the highest recorded in peanuts seeds followed by Aspergillus section Nigri and Aspergillus section Circumdati was the least 34 . Past study by Tournas et al 5 found the same results that A. niger followed by Penicillium were the most common mold in nuts and dried fruits. Rhizopus stolonifer was isolated from peanuts, almonds and raisins in high and moderate frequency of occurrence (Table 1). In a study by Abdulla 35 , reported that Aspergillus, Rhizopus and Penicillium genera were more frequently detected than other genera of fungi in nuts.
The molecular identification of the tested A. niger confirmed the morphological identity and more similarity among A. niger isolates was observed and low similarity with A. niger AHBR5 except A. niger-27 (Figs. 1, 2) and the obtained results in agreement with Perrone et al 36,37 . All the tested A. niger isolates were ochratoxin producers with variable readings by using fluorometric method ( Table 2). In past investigation by Al-Sheikh 38 confirmed that 57% and 60% of A. niger and A. carbonarius, respectively deteriorated peanut were ochratoxin producers. Magonli et al 39 , demonstrated that 32% of Aspergillus section Nigri obtained from peanut seeds in Argentinean had ability to produce ochratoxin A. Alhussaini 30 , found that 33.3% of Aspergillus section Nigri biserriate and one isolate of uniserriate isolated from nuts were ochratoxin A producers. The tested A. niger isolates recovered from baby foods recorded positive results for ochratoxins production 40 . Our obtained results were in-disagreement with past study by Palumbo & O'Keeffe 41 , reported that all the tested 171 isolates of Aspergillus section Nigri isolated from almonds showed negative results for ochratoxin A production. In this study, ochratoxin biosynthesis gene Aopks was detected in all the tested isolates at 549 bp (Fig. 3). The obtained results in harmony with Massi et al. 42 , who reported that pks gene was detected in all Aspergillus niger positive ochratoxgenic strains isolated from Brazilian foods amongst, nuts and dried fruits. Aopks genes were detected at 549 bp in A. niger isolates that had ability to produce ochratoxins 43,44 . All tested A. niger and A. tubingensis isolated from beef showed positive results for the presence of pks genes 45 . The selected A. niger isolates were lipase producers qualitatively on Tween 80 solid medium and quantitatively by using trimetric titration method with the highest activity recorded by A. niger-26 isolated from almond (Table 3). A. niger is well-recognized to be the best producer of lipase enzyme and is favored in many industrial processes 46,47 . A. niger, Fusarium oxysporum and Nectria haematococca isolated    www.nature.com/scientificreports/ from beef luncheon were the highest lipase producers 48 . Rai et al. 49 , isolated lipase producer A. niger from some oil contaminated soil samples. Earlier studies also, confirmed that A. niger was the highest lipase producing strain [50][51][52][53][54] . Putri et al. 12 , optimized the production of lipase by A. niger by using agro-waste and revealed that 1% olive oil was the highest inducer, yielding dry lipase extract with highest activity unit (176 U/ml enzyme).
Lip2 gene was visualized at 1276 bp in all the tested A. niger isolates (Fig. 4). Yang et al. 55 , reported Lip2 gene a novel lipase gene cloned from A. niger. Lipase exhibits antibacterial activity against Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa with MBC of 20 µl/100 µl (Table 4). This may be due to that lipase acting on the lipopolysaccharide of Gram negative cell wall as well as the esters of exopolysaccharide present in the biofilm. Furthermore, lipolytic enzyme acts on a lipid substrate Such as phospholipids and other hydrophobic molecules, to hydrolyze or esterify a bond 16 . Lipases are esterases capable of hydrolyzing any ester bond. They act on the lipoprotein, lipopolysaccharide and phospholipids which surrounds the peptidoglycan layer leading to the hydrolysis of the lipid bilayer. The lipopolysaccharide complex is an endotoxin present on the outer membrane of the cell wall and this toxicity leads to a wide spectrum of nonspecific pathophysiological reactions including fever, changes in white blood cell counts, disseminated intravascular coagulation, hypotension, shock and death. When lipase works on lipid A, the chances of infection are reduced 56 . In most of the Gram positive bacteria, lipoteichoic acids are present and the lipid tail present here plays a major role in the bacterial attachment. There is a possibility for the lipase to act on this lipid tail thereby preventing its adherence to a surface 16 . Our results confirmed antibacterial activity of lipase on Gram positive bacteria (MRSA) with MBC of 40 µl/100 µl (Table 4). Bacterial biofilms pose a great threat to human life not only because they involved in a lot of chronic infectious human diseases but also, they highly resistant to different antimicrobial agents. This generates a strong demand for finding suitable anti biofilm agents 57 . Bacterial biofilms are common populations of bacterial cells surrounded by a self-produced matrix of extracellular polymeric substances (EPS) 58 . EPS surrounding mixture include various exopolysaccharides, lipids, secreted proteins some of which can form amyloid fibers and extracellular DNA 59 .
Most of the antimicrobial agents fail to penetrate the biofilm owing to the presence of EPS which acts as a barrier protecting the bacterial cells within the biofilm. So, the remedy will be the use of compounds that able to degrade the biofilm EPS. Enzymes have been recognized to be effective for the degradation of the biofilms EPS 60,61 . Plants contain various anti-biofilm compounds, as they have to prevent bacterial growth on their surfaces 62,63 . Since lipase, an esterase, is a hydrolyzing enzyme, it is having the ability to act on the EPS produced by the organisms 64 , by degrading protein components and the high molecular weight lipid of the biofilm 65 . In the current investigation crude lipase, was examined as antibiofilm agent against four human pathogens by spectrophotometric methods. The highest significant percentages of inhibition were 95.3, 74.9, 77.1 and 93.6 for Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis and Methicillin-resistant Staphylococcus aureus (MRSA), respectively. Although, all added volumes of lipase significantly inhibited biofilm formation, the suitable volume that gives highest inhibition percentage was 50 µl (Fig. 5). Scanning electron microscopy (SEM) has been used widely for qualitative observation of biofilm before and after treatments, biofilm disturbance due to its high resolution and www.nature.com/scientificreports/ is usually applied in biological assays of biofilm removal effectiveness also antimicrobial treatments [66][67][68] . Results of SEM confirmed antbacterial and antibiofilm properties of crude lipase against the tested human pathogens.
In conclusion, Peanuts were the highest contaminated samples among the tested types. A. niger was the most isolated species from nuts. All the selected A. niger isolates were lipase producers with highest enzyme activity was recorded by A. niger MW029470 and showed positive results for the presence of Lip 2 gene. Crude lipase from A. niger MW029470 showed highly inhibition of the tested pathogens growth with MBC of 20 to 40 µl/100 µl and significantly inhibited biofilm formation of 4 biofilm former human bacterial pathogens. The significant percentages of inhibition were 95.3, 74.9, 77.1, and 93.6 for Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis and Methicillin-resistant Staphylococcus aureus (MRSA), respectively.

Materials and methods
Collection of nuts samples. Eighty samples of peanut, almond, coconut and raisin (20 samples of each type) were purchased from different supermarkets at Qena Governorate, Egypt. All samples were kept in a refrigerator until mycological analysis.
Isolation of fungi. The modified method described by Tournas et al. 5 was employed for isolation of mycobiota contaminating nuts. A known weight of each sample was blended with 90 ml of 0.1% peptone in blender jar under aseptic conditions for minute. Serial dilutions were made to obtain the suitable one. One ml of the suitable dilution was poured in sterilized petri plate followed by 20 ml of rose Bengal chloramphenicol agar (RBCA) medium containing g/l (peptone; 5, glucose; 10, kH 2 PO 4 ; 1, MgSO 4 . 7H 2 O; 0.5, rose Bengal; 0.05, chloramphenicol; 0.1, and agar 15.5). Triplicates of each sample were prepared. Plates were incubated for a week at 28 °C. The developed fungal colonies were counted, examined and identified (based on macro-and microscopic features) 69 .

Sequence analysis of 5.8S-ITS region. ITS1 and ITS2 regions together with 5.8S gene in rDNA from
A. niger strains were amplified as designed by Hermosa et al. 70 . The purified bands were determined using the sequencer Gene analyzer 3121 in Scientific Research Center, Biotechnology and Genetic Engineering Unit, Taif University, KSA. The realized sequence was aligned using Molecular Evolutionary Genetics Analysis (MEGA) version 5.10. Then, a consensus sequence was generated from each alignment made. The sequencing data were compared against the Gene Bank database (http:// www. ncbi. nlm. nih. gov/ BLAST/), where a nucleotide blast program was chosen to identify the homology between the PCR fragments and the sequences on the Gene Bank database.

MW029474.
Ochratoxins production by A. niger isolates. Five isolates of Aspergillus niger with the highest number of colonies and high frequency of occurrence were tested for their ability to produce ochratoxins by cultivation in conical flasks containing 50 ml of yeast extract sucrose (YES) liquid medium with composition sucrose, 40 g, yeast extract 20 g, and distilled water, 1000 m1. Incubation the flasks at 28 °C for fifteen days 71 . Filtration through a fluted filter paper (Whatman 2 V, Whatmanplc, Middlesex,UK). Total ochratoxins were determined according to the method mentioned by El-Dawy et al. 44 in 10 ml fungal filtrate by adding 90 ml (methanol: water) (80:20 v/v) and the filtrate was diluted (1:4) with distilled water and re-filtered through a glass-fiber filter paper. Ten milliliters of the glass-fiber filtrate were placed on Ochra test WB SR Column (VICAM, Watertown, MA, USA) and allowed to elute at 1-2 drops/s. The columns were washed twice with 10 ml of distilled water, and ochratoxins were eluted from the column by adding 1 ml of methanol HPLC and delivered in clean cuvette. 1.5 ml ochratoxin eluting agent was added and the total ochratoxins concentration were measured after calibration VICAMSeries-4 fluorometer set at 360 nm excitation and 450 nm emissions 72 .
Molecular detection of ochratoxin-producing genes. DNA extraction and purification were performed using DNA Promega Kit DNeasy Blood & Tissue (Valencia, CA, USA). Two published primers were used for the specific detection of ochratoxin biosynthesis genes. The sequence of primers was as following: Aopks-F '5-CAG ACC ATC GAC ACT GCA TGC-'3, Aopks-R '5-CTG GCG TTC CAG TAC CAT GAG-'3 73 . The 630 bp fragments were amplified, PCR was performed in a reaction volume of 25 μl according to Hussein et al. 74 The reactions were done in a C1000.
Thermo Cycler BioRad, Germany with initial denaturizing at 94 °C for 5 min, followed by 30 cycles of 1 min. at 94 °C, 1 min. at 58 °C and extension at 72 °C for 1 min 43 , then final step as extension at 72 °C for 10 min. PCR products were checked on a 1.3% agarose gel and stained with ethidium bromide.
Screening A. niger isolates for lipase production. Tween 80 agar plate was used for screening the tested isolates for lipase production containing (g/l peptone, 15; NaCl, 5; CaCl 2 , 1; tween 80, 10 and agar, 15) and pH of the media was adjusted to 7. 250 µl of fungal spore suspension (8 × 10 7 spores/ ml) was inoculated to 8 mm cavity on the media and incubated at 28 °C for 4 days. Appearance of white precipitate around the fungal colony indicates the ability to produce lipase enzyme 49 . Quantitative estimation of lipase. Trimetric method was applied for assay lipase 75 with some modification. Two disks (8 mm) of tested isolates were inoculated to minimal medium containing (g/l 1 yeast extract, www.nature.com/scientificreports/ tor at 150 rpm for 3 days. 1.5 ml suspension from liquid medium was centrifuged at 3000 rpm for 10 min. Assay mixture containing 500 µl 0.1 M phosphate buffer (pH 6.8), 500 µl tween 80 and 250 µl crude enzyme was incubated at 37 °C for 20 min. Three ml of acetone: ethanol (1:1) was added to stop the reaction. The liberated fatty acids were titrated with 1 N NaOH solution with bromothymol blue indicator. In control 250 µl distilled water was added instead of crude enzyme. Lipase activity (U/ml-min) was calculated from the following equation: T is the titration volume, C is the control, N is the normality of NaOH, df is the volume of assay/volume of enzyme, t is the incubation time, v is the sample.
Detection of lip2 gene in Aspergillus niger isolates. Aspergillus niger lip2 gene was detected by using 2 documented primers. The sequences of primers were as following: P1 (5'-CTC AAG AGT ATC CTG CAC TG-3') and P2 (5'-CTG AAC CTT CCT TGG GAT AG-3') 55 . Twenty-five μl as volume was used for PCR reaction by mixing 12.5 µl of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 µl of each primer, 6 µl of DNA template and 4.5 µl of water was added to make the volume up to 25 μl. Applied biosystem 2720 thermal cycler was used for performing the reaction with initial denaturizing at 94 °C for 5 min, followed by 35 cycles of 30 s. at 94 °C, 50 s. as annealing temperature at 59 °C and 1 s. at 72 °C. Ten minutes at 72 °C was used as the final extension. The PCR products were checked on 1.5% agarose gel in 1 × TBE buffer. A gelpilot 100 bp plus DNA Ladder was used to determine the fragment sizes. The gel was photographed by a gel documentation system. Data was analyzed through computer software.

Determination of minimum bactericidal concentration (MBC).
The bactericidal efficacy was defined as a 99.9% decrease in CFU (3 logs) in the initial inoculum during 24 h of incubation. The MBC was determined by inoculating sterilized tryptic soy agar (TSA) fresh plates with 50 µl from each well of overnight MIC plates. Viable colonies were counted after 24 h at 37 °C. The limit of detection for this assay was 10 cfu ml −162,79 .
Static biofilm assay. The tested bacterial strains {Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis and Methicillin-resistant Staphylococcus aureus (MRSA)} were obtained kindly from international Luxor hospital. The ability of the verified pathogens for biofilm formation was determined using 96-well polystyrene plates 80 . Bacterial strains were subcultured on tryptic soy agar for 24 h at 37 °C, suspended in tryptic soy broth and adjusted to an OD 595 of 0.02. 130 µl of each adjusted isolate culture were put in the microtitre plate (U bottom, Sterilin) at 37 °C for 24 h. After incubation the wells were washed six times with distilled water, Furthermore, the wells were stained with 0.1% crystal violet for 10 min. the wells were again washed with distilled water (4 times) to remove excess stain 81 . Finally, the wells were destained by 210 µl of ethanol 96% and the OD 595 was read using infiniteF50 Robotic (Ostrich) microplate plate to quantify the amount of biofilm.
Antibiofilm efficacy of crude lipase. The effect of crude lipase enzyme with the highest activity from A. niger MW029470 free of ochratoxin after 3 days of incubation as antibiofilm against four human pathogenic biofilm former bacteria was done by spectrophotometric methods. Different volumes (20,30,40,50,60, and 70 µl) were added to 130 µl of the tested pathogens at OD 595 of 0.02 after 24 h incubation at 37 °C for allowing biofilm formation. The plates then incubated for further 24 h and then stained with crystal violet as described previously 27 .
Scanning electron microscopy (SEM) analysis of antibacterial and antibiofilm efficacy of crude lipase. Preparation of samples for antibacterial was performed as described by Wang et al. 82 . While for biofilm was done as described by Kong et al. 83 and Chin et al. 84 with little modifications. Biofilms were allowed to form on the slides at 37 °C for 24 h alone (control) and after treatment with crude lipase following which, the samples were fixed in 4% (v/v) glutaraldehyde in 0.05 M phosphate buffer (pH 7.0) at 4 °C for 12 h. Subsequently, the samples were washed three times in phosphate buffer, dehydrated through a graded ethanol series, dried in a critical-point drying apparatus with liquid carbon dioxide; slides coated with gold and viewed using (JEOL JSM-5500LV, Japan). Statistical analysis. The variability degree of results was expressed in form of means ± standard deviation (mean ± SD) based on triplicates determinations (n = 3 for replicate plates). The data were statistically analyzed by one-way ANOVA analysis and compared using the least significant difference (LSD) test at 0.05 (*) levels. It was done to compare between control and treatments.