Optimizing and purifying extracellular amylase from soil bacteria to inhibit clinical biofilm-forming bacteria

Background Bacterial biofilms have become a major threat to human health. The objective of this study was to isolate amylase-producing bacteria from soil to determine the overall inhibition of certain pathogenic bacterial biofilms. Methods We used serial dilution and the streaking method to obtain a total of 75 positive amylase isolates. The starch-agar plate method was used to screen the amylolytic activities of these isolates, and we used morphological and biochemical methods to characterize the isolates. Optimal conditions for amylase production and purification using Sephadex G-200 and SDS-PAGE were monitored. We screened these isolates’ antagonistic activities and the purified amylase against pathogenic and multi-drug-resistant human bacteria using the agar disk diffusion method. Some standard antibiotics were controlled according to their degree of sensitivity. Finally, we used spectrophotometric methods to screen the antibiofilm 24 and 48 h after application of filtering and purifying enzymes in order to determine its efficacy at human pathogenic bacteria. Results The isolated Bacillus species were Bacillus megaterium (26.7%), Bacillus subtilis (16%), Bacillus cereus (13.3%), Bacillus thuringiesis (10.7%), Bacillus lentus (10.7%), Bacillus mycoides (5.3%), Bacillus alvei (5.3%), Bacillus polymyxa (4%), Bacillus circulans (4%), and Micrococcus roseus (4%). Interestingly, all isolates showed a high antagonism to target pathogens. B. alevi had the highest recorded activity (48 mm) and B. polymyxa had the lowest recorded activity (12 mm) against Staphylococcus aureus (MRSA) and Escherichia coli, respectively. On the other hand, we detected no antibacterial activity for purified amylase. The supernatant of the isolated amylase-producing bacteria and its purified amylase showed significant inhibition for biofilm: 93.7% and 78.8%, respectively. This suggests that supernatant and purified amylase may be effective for clinical and environmental biofilm control. Discussion Our results showed that soil bacterial isolates such as Bacillus sp. supernatant and its purified amylase are good antibiofilm tools that can inhibit multidrug-resistant former strains. They could be beneficial for pharmaceutical use. While purified amylase was effective as an antibiofilm, the isolated supernatant showed better results.

INTRODUCTION enrichment broth. Samples were incubated at 37 • C for 24 h. The landowner, Mohamed El sanousy, approved field sampling.

Screening and isolation of amylase-producing bacteria
Serial dilution techniques are one of the most precise methods for isolating bacteria from soil (Jamil et al., 2007;Rasooli et al., 2008). We performed serial dilutions up to 10 −7 . We aseptically transferred 100 µl from each dilution, which we spread into tryptic soy agar media fortified with 1% starch. The plates were incubated at 37 • C for 24 h to determine the colony-forming unit (CFU)/ml. The plates were then flooded with iodine that turns blue when it reacts to unhydrolyzed starch. If the starch was hydrolyzed, a clear halo zone would appear against a dark blue background around the colonies that produce amylase (Gupta et al., 2003;Abd-Elhahlem et al., 2015). We further subcultured bacterial isolates to obtain a pure culture and identified isolates using standard morphological techniques based on colony shape, Gram's staining, spore formation, and biochemical characterization (Cruickshank et al., 1975;Collins & Lyne, 1984;Koneman et al., 1992). Isolates were then maintained in a 70% sterilized glycerol stock at −70 • C for further use.

Selecting isolates for amylase purification
We selected isolates for amylase extraction and purification, as well as for comparing the purified amylase's antibiofilm activity against some human pathogenic bacteria, according to the starch hydrolysis ratio (SHR) that we calculated using the following equation (Pranay et al., 2019): SHR = clear halozone diameter (mm)/colony growth diameter (mm).
Isolates were subcultured on starch agar plates, which were incubated for 24 h at 37 • C. After incubation, the plates were flooded with iodine. Finally, we calculated SHR using the equation above.

Effect of temperature and incubation periods
The starch nutrient medium was prepared and the pH was adjusted to 7.5. We then inoculated the medium with the tested isolates. The culture was allowed to grow on a rotatory shaker (250 revs/min) at temperatures ranging from 15 to 65 • C over 48 h. We took 20 ml from each culture at all temperatures and time intervals (18, 24, 48, 72, 96, and 120 h) and centrifuged them to remove the bacterial cells. Finally, the supernatant was collected to assay the amylase activity (Nimisha, Moksha & Gangawane, 2019).

Effect of pH
We prepared the starch nutrient medium and adjusted the pH to different values (5, 6, 7, 8, 9, and 10). Each isolate was inoculated into a portion of this medium and were grown at 50 • C for 24 h. We then collected 20 ml from each isolate and applied the same treatment as above to determine amylase activity (Nimisha, Moksha & Gangawane, 2019).

Effect of starch concentration
All Bacillus isolates were grown on nutrient broth medium with a pH of 9, except B. subtilis which was grown at a pH of 7. Different soluble starch quantities were added to fresh medium to give final concentrations of 0.1, 0.5, 1, 1.5, 2, 2.5, and 3%. We inoculated each isolate in this medium at 50 • C for 24 h to determine their amylase activity (Nimisha, Moksha & Gangawane, 2019).

Determining amylase activity under optimum conditions
The assay mixture contained 2 ml of a solution made up of 1% starch in 50 mM sodium phosphate buffer (pH 7) and 0.1 ml of enzyme solution. After 10 min. of incubation at 40 • C, we stopped the reaction by adding 2 ml of 3,5 dinitrosalicylic acid (DNS) reagent, and heated the tubes at 100 • C for 5 min. The absorbance was measured spectrophotometrically at 540 nm using a blank containing buffer instead of the culture supernatant. We calculated the amount of reduced sugars from a maltose standard curve (Meyer, Fisher & Bernfeld, 1951). Protein was determined using Bradford's (1976) method.

Ammonium sulfate precipitation
The crude amylase enzyme was brought to 45% saturation with ammonium sulfate and was kept overnight in a cold room at 4 • C. We removed the precipitate, brought the supernatant to 85% saturation with ammonium sulfate, and centrifuged it at 8,000 rpm for 10 min at 4 • C. After collecting the precipitate during this step, we stored it at 4 • C (Shinde & Soni, 2014).

Dialysis
This step was conducted to exclude the ammonium sulfate remains and to concentrate the enzyme. We used the dialysis tubes, which were previously soaked in 0.1 M phosphate buffer (pH 6.2), for precipitate dialysis. The precipitate was dissolved in 0.1 M phosphate buffer and was dialyzed against the same buffer (Roe, 2001).

DEAE Sephadex G-200
The crude enzyme preparations of the six culture filtrates were applied separately to a column of DEAE-Sephadex G-200. The enzyme was eluted with a linear gradient of sodium chloride (0 -0.4 M) in 200 ml of sodium phosphate buffer (0.05 M and pH 7), the flow rate was adjusted to 1 ml per 1 min., and 200 ml of eluents were collected into 40 tubes (1 × 7 cm) using an automatic circular fraction collector. We determined enzyme activity and protein concentration in each fraction using the described assay method. Fractions of the highest specific activity were pooled together and kept for further study.

SDS-PAGE
We carried out polyacrylamide gel electrophoresis according to Laemmli's (1970) method using 10% polyacrylamide gel. Purified B. alvei and B. cereus amylase was loaded into wells parallel to the standard protein markers. The protein bands were stained with Coomassie brilliant blue (Sigma, St. Louis, MO, USA). We estimated the enzyme's relative molecular weight by comparing it to molecular mass standard markers (Fermentas, Vilnius, Lithuania).

Antibacterial activity of purified amylase enzyme from the isolated Bacillus
We placed 100 µl of purified amylase from the selected isolates according to their SHR in the wells of the agar plates inoculated with the target strains. The plates were incubated at 37 • C for 24 h. The halo zone was measured using a ruler.

Biofilm formation assay
We determined the biofilm formation ability of the tested pathogens (E. coli, P. aeruginosa, S. aureus (MRSA), K. pneumoniae, and A. baumanii) using 96-well polystyrene plates (Seper et al., 2011) and the methods described by Salem et al. (2015a) andSalem et al. (2015b): isolates 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. We placed 130 µl of each adjusted isolate culture in the microtitre plate (U bottom, Sterilin) for 24 and 48 h at 37 • C. After incubation, the wells were washed with distilled water (six times) and were stained with 0.1% crystal violet for 10 min. The wells were then washed again with distilled water (four times) to remove excess stain. Finally, the wells were destained using 210 µl of ethanol 96%, and the OD 595 was read using an Infinite R F50 Robotic (Ostrich) Microplate Plate to quantify the amount of biofilm.

Antibiofilm activity of the isolated Bacillus sp. filtrate and its purified amylase enzyme
We compared the antibiofilm effects of the isolated bacteria filtrate and the purified amylase from the selected isolates against the five human biofilm pathogenic bacteria using the following spectrophotometric methods: a fresh isolate culture was prepared and adjusted to 0.5 McFarland (10 6 CFU/ml), and 30 µl (this volume was selected according to a preliminary experiment) of these cultures and purified amylase enzyme were added to 130 µl of the tested pathogens at an OD 595 of 0.02 after 24 h of incubation at 37 • C to allow biofilm formation. The plates were then incubated for 24 and 48 h and stained with crystal violet. Wells without isolated cultures or amylase served as controls.

Statistical analysis
The variability degree of the results was expressed in the form of mean ± standard deviation (mean ±SD) based on three independent determinations (n = 3). We statistically analyzed the data by one-way ANOVA analysis and compared the control and treatment groups using the least significant difference (LSD) test at 1% (*) levels (Snedeco & Cochran, 1980).

Screening and isolating amylase-producing bacteria
Microorganisms that produce amylase are generally isolated from soil and other sources (Fossi, Taveaand & Ndjonenkeu, 2005). Our study explored the isolation of amylaseproducing bacteria from soil using the serial dilution spread plate technique. Singh & Kumari (2016) used a similar method by diluting soil samples on starch agar plates and flooding the plates with an iodine solution. The presence of a halo zone around certain colonies indicated amylase production, and a total of 75 bacterial isolates showed a zone of clearance with a starch agar medium. Bacterial isolates were selected according to their amylolytic activity (Table 1). A similar method was also employed by Magalhaes (2010). We further characterized isolates using morphological and biochemical tests shown in Tables

Effect of temperature and time intervals
All isolates showed maximum amylase production after 24 h. Similar results were obtained by Singh & Kumari (2016), who observed that the highest amylase activity of some Bacillus sp. occurred at 24 h of incubation and that the activity began to decrease after 48 and 72 h of incubation time (Fig. 1B). B. megaterium, B. subtilis, and B. cereus showed maximum amylase production at 45 • C, while other isolates showed maximum amylase production at 55 • C. Mohamed, Malki & Kumosani (2009) similarly reported that some amylase were stable at 40 • C and some at 50 • C (Fig. 1A).

Effect of pH
All Bacillus isolates showed maximum amylase production at a pH of 8, except for B. subtilis which maximally produced amylase at a pH of 7. A previous study by Behal et al. (2006) found that the optimum pH for amylase production was 8. Another study by Singh & Kumari (2016) reported that while amylase activity was recorded at different pH levels from 5 to 10, maximum activity was observed at pH 7 (Fig. 1C).

Effect of substrate concentration
Our results showed that B. subtilis and B. cereus had maximum amylase production at 1.5% soluble starch concentration. The remaining isolates showed maximum amylase production at 2.0% soluble starch concetration (Fig. 1D). Mishra & Behera (2008) reported that Bacillus strains produced the maximum yield of amylase at a starch concentration of 2%.

Enzyme activity
We purified extracellular amylase from the Bacillus isolated from soil to homogeneity using 45-85% ammonium sulfate precipitation and Sephadex G-200 (Fig. 2). As shown in Table 3

SDS-PAGE
After purification, the SDS-PAGE profile showed a single protein band of amylase for each bacteria, confirming that the enzyme has been purified to homogeneity. The molecular weights of B. alvei and B. cereus were 60 KDa and 43 KDa, respectively (Fig. 3). The molecular weight of B. alvei was similar to that of the amylase isolated from B. subtilis (56 KDa and 55 KDa, respectively) (Bano et al., 2011;Takkinen et al., 1983). The molecular weight of B. cereus was equal to the molecular weight of amylase from B. cereus and B. subtilis (42 KDa) (Annamalai et al., 2011;Das, Doley & Mukherjee, 2004). Previous studies have reported different molecular weights for amylase isolated from Bacillus sp. Lin, Chyau & Hsu (1998) found that Bacillus sp. can produce five different forms of amylase. Antibacterial activity

Antagonistic efficacy of the isolated bacteria and purified amylase enzyme from selected isolates
In this study, we compared the antimicrobial activity of Bacillus supernatant and purified amylase with standard antibiotics against human pathogens (Table 4). The standard antibiotics served as the control group since pathogenic bacteria can become extremely resistant to widely-used antibiotics. The pharmaceutical industry is in need of new and natural antimicrobials that can overcome the problem of multidrug-resistant strains (Schmidt, 2004;Salem et al., 2015a;Salem et al., 2015b;Salem et al., 2017). Several soil organisms can produce antibiotics using a survival mechanism that can eliminate their competition (Talaro & Talaro, 1996;Jensen & Wright, 1997). The Bacillus genus is a terrestrial strain that can produce inhibitory compounds from peptide-derivative and  lipopolypeptide antibiotics (Mannanov & Sattarova, 2001;Tamehiro et al., 2002;Stein, 2005). Oscariz, Lasa & Pisabarro (1999) and Yilmaza, Sorana & Beyatlib (2006) found that isolated bacteriocin-producing strains such as Bacillus sp. were active against gram-negative and gram-positive bacteria. We compared the antimicrobial activity of the isolated amylaseproducing bacteria and purified amylase against five human pathogenic bacteria (Table 4). We found that E. coli was resistant to sulfamethoxazole-trimethoprim (23.75/1.25 mcg), gentamycin (10 µg), cefotaxime (30 µg), piperacillin (100 µg), and piperacillin-tazobactam (100/10 µg); showed intermediate sensitivity to ampicillin-sulbactam (10/10 mcg); and was sensitive to chloramphenicol (30 µg) and meropenem (10 µg Perez, Suarez & Castro (1992) and Aslim, Saglam & Beyatli (2002) found that B. subtilis, B. thuringiesis, and B. megaterium showed antibacterial activity against E. coli and P. aeruginosa. We found that S. aureus (MRSA) was resistant to oxacillin (1 mcg), vancomycin (30 mcg), penicillin G (10 U), cefotaxime (30 mcg), and gentamycin (10 µg), and showed intermediate sensitivity to chloramphenicol (30 µg), erythromycin (15 mcg), and sulfamethoxazole-trimethoprim (23.75/1.25 µg). The isolated amylase-producing bacteria showed better antibacterial effects on the tested pathogens, with the greatest effect shown by B. alvei (48 mm) and the least effect shown by B. cereus (14 mm) (Table 4). However, B. mycoides and M. roseus did not affect S. aureus. Similar results were obtained by Moshafi et al. (2011) andRamachandran et al. (2014). In contrast to the high antimicrobial activity observed in the isolated soil bacteria, the purified amylase from the selected isolates had very little effect on E. coli and K. pneumoniae (the highest inhibition diameter was 7.5 mm), and no recorded effect in response to the other tested pathogens (Table 4). This result is similar to that of Kalpana, Aarthy & Pandian (2012), who confirmed that amylase enzyme has no antibacterial effect.

Biofilm formation assay
We quantitatively determined the amount of biofilm (OD 595 ) in the tested pathogens and designated the 24 and 48 h treatments as the control groups (Figs. 4, 5, 6, and 7). Using the OD 595 nm mean values, we defined the pathogens as low, moderate, or high bacterial biofilm formers when the OD 595 nm was > 1, 1 -2.9, and < 2.9, respectively. A. baumanii and Klebsiella pneumoniae were high biofilm formers while E. coli, P. aeruginosa, and S. aureus (MRSA) were low biofilm formers.

Antibiofilm activity of isolated bacterial filtrate and purified amylase enzyme from selected Bacillus isolates
In a natural ecosystem, bacteria can be exist in two forms: planktonic cells, which are susceptible to antibiotics and other antimicrobial agents, and biofilm, which are resistant to antibiotics and disinfectants (Limoli, Jones & Wozniak, 2015). A biofilm is a complex community of bacteria attached to a surface or interface enclosed in an exopolysaccharide

Inhibition zone in mm b
Bacillus megaterium 21 ± 1.5 26 ± 1 36 ± 1 24 ± 1 31 ± 1 Bacillus subtilis 12 ± 1 18 ± 2 21 ± 1 15 ± 1.5 20 ± 2 Bacillus cereus 12 ± 1.2 21 ± 1 36 ± 1 18 ± 1 14 ± 1.5 Bacillus thuringiesis 14 ± 1.5 21 ± 1 21 ± matrix, protected from unfavorable antibiotics, host defenses, or oxidative stresses (Shakibaie, 2018). Microbial biofilms have created huge problems in the treatment of both community and hospital infections. Most antimicrobial agents are unable to penetrate biofilm due to its extracellular polymeric substances (EPS), which act as a barrier protecting the bacterial cells within the biofilm. Therefore, we must use compounds that have the potential to degrade the biofilm's EPS. Enzymes have proven to be effective in EPS degradation (Kalpana, Aarthy & Pandian, 2012;Lequette et al., 2010). In our study, we compared the antibiofilm activity of the Bacillus sp. that we isolated from soil (supernatant) and the purified amylase from these isolates against five human pathogenic biofilm former strains. Our study has reported that Bacillus supernatant and amylase enzyme can inhibit the biofilm formation in various pathogens. We confirmed the ability of pathogenic bacterial strains to form biofilm formation using spectrophotometric methods before applying the antibiofilm treatments of bacterial filtrate and purified amylase enzyme (Figs. 4, 5, 6, and 7). The antibiofilm activity was screened using a spectrophotometric method with crystal violet staining. Our results showed that the bacteria isolated from soil exhibited significant antibiolfilm effects against the tested pathogenic strains after 24 h of treatment. The percentage of inhibition significantly increased after 48 h of treatment. The highest percentage of inhibition was recorded for B. circulans against K. pneumonia: 93.7% after 48 h of treatment (Fig. 5D, T8). We also monitored the efficacy of the purified amylase enzyme as an antibiofilm against the same tested pathogens. Our results revealed that the purified amylase showed significant antibiofilm effects after 24 h of treatment.    Vaikundamoorthya et al. (2018) confirmed the antibiofilm efficacy of the thermostable amylase enzyme from B. cereus. It is worth noting that the isolated bacteria filtrate showed great antibiofilm activity compared to the purified amylase enzyme from the selected isolates. This may be due to the accumulation of some extracellular and intracellular metabolites in the medium, which is further explained by the metabolic overflow theory (Pinu, Villas-Boas & Aggio, 2017;Pinu et al., 2018;Horak et al., 2019). Bacillus also showed great efficacy in the production of carbohydrate-active enzymes and bioactive compounds, as well as the secretion of a variety of extracellular metabolites and lytic enzymes (Abdel-Aziz, 2013). Additionally, Bacillus species are the most efficient at producing peptide antibiotic compounds such as polymyxin, colistin, and circulin (Katz & Demain, 1997;Atanasova-Pancevska et al., 2016). Our results also indicated a great inhibition of biofilm from the amylase enzymes of B. alvei (96.02 U/ml), followed by B. thuringiesis (88.64 U/ml), B. megaterium (80.03 U/ml), B. subtilis (76.0 U/ml), B. cereus (55.9 U/ml), and B. lentus (45.69 U/ml) (Table 3). This may be due to the increased enzyme activity in each species.

CONCLUSION
Our results indicated that the ability of Bacillus sp. to produce extracellular and intracellular metabolites, lytic enzymes, and some peptide antibiotics directly affects the antimicrobial functions of various Bacillus sp. (amylase producers) in the soil. We observed the highest inhibition rate (93.7%) when comparing the species' antibiofilm effects against five human pathogenic strains. We observed an inhibition rate of 78.8% when comparing the antibiotic biofilm activity of purified amylase against the strains. Our study showed that Bacillus filtrate is an effective clinical antibiofilm. Futher studies are being conducted to determine the exact composition of the filtrate and its active agents.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
The authors received no funding for this work.