International Journal of Biological Macromolecules 115 (2018) 762–766
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The role of probiotic Lactobacillus acidophilus ATCC 4356 bacteriocin on effect of HBsu on planktonic cells and biofilm formation of Bacillus subtilis Maliheh Sarikhani a, Rouha Kasra Kermanshahi a,⁎, Parinaz Ghadam b, Sara Gharavi b a b
Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 4 May 2017 Received in revised form 10 January 2018 Accepted 18 March 2018 Available online 20 March 2018 Keywords: Bacillus subtilis BM19 HBsu Lactobacillus acidophilus ATCC 4356
a b s t r a c t Bacillus subtilis is a Gram positive, aerobic and motile bacterium. Biofilm formation is an important feature of this bacterium which confers resistance to antimicrobial agents. The use of new antimicrobial reagents which eliminate biofilms are important and necessary. In this study, the effect of secondary metabolites (bacteriocin) from Lactobacillus acidophilus ATCC 4356 on Bacillus subtilis BM19 in the presence and absence of HBsu which is involved in the growth of planktonic cells and biofilm formation, is reported. HBsu nucleoprotein plays several roles in different processes of Bacillus subtilis cells such as replication, transcription, cell division, recombination and repair. In this study, for the first time, the effect of HBsu on biofilm formation is presented. Results: In the absence of HBsu, purified bacteriocin from L. acidophilus ATCC 4356 was more effective in inhibiting growth of B. subtilis BM19 planktonic cells as well as biofilm formation. The presence of HBsu on the other hand led to increased biofilm formation. © 2018 Published by Elsevier B.V.
1. Introduction
2. Materials and methods
Probiotics and their secondary metabolites, bacteriocins, are appropriate supersedes of antibiotics because of their safe antibacterial effects [1]. Lactobacillus acidophilus is a Gram-positive, and nonspore forming bacterium [2]. Certain strains of lactic acid bacteria produce bacteriocins which are proteinaceous compounds with widespread spectrum of antimicrobial effects [2]. Bacteriocins are synthesized by Gram positive or Gram negative bacteria [3]. The bacteriocins produced by lactic acid bacteria are small and ribosomally synthesized, antimicrobial peptides or proteins with activity against closely-related Gram-positive bacteria, whereas producer cells are immune to their own bacteriocin. Bacillus subtilis is a gram positive and endospore-forming bacterium [4]. In the strains of Bacillus subtilis, HU nucleoprotein with different roles in cell function is named HBsu [5]. Biofilm formation is another feature of Bacillus subtilis which creates resistance of this bacterium against antimicrobial agents and environmental menances [6]. HU nucleoprotein is bound to DNA in a non-specific manner; the absence of this protein causes disintegration of biofilm in Streptococcus [7,8]. For the first time in this study, the role of HBsu in biofilm formation of B. subtilis and the importance of HBsu presence in the effect of bacteriocins on B. subtilis growth is presented.
2.1. Bacterial strains and culture conditions
⁎ Corresponding author. E-mail address:
[email protected] (R.K. Kermanshahi).
https://doi.org/10.1016/j.ijbiomac.2018.03.087 0141-8130/© 2018 Published by Elsevier B.V.
Lactobacillus acidophilus (ATCC 4356) was purchased from Iranian Research Organization, Bacillus subtilis (BM19) and Bacillus subtilis (wild type) were the kind gifts from Prof. Dr. Mohamed A. Marahiel at Philips University for Science and Technology (Germany). In order to study hbs as an essential gene, B. subtilis BM19 strain was constructed, containing a truncated copy of the gene downstream of its own promoter and another intact copy under control of the isopropyl-betaD-thiogalactopyranoside (IPTG)-inducible spac-1 promoter [5]. Lactobacillus acidophilus was grown in Man-Rogosa-Sharpe broth (MRSB; Merck, Darmstadt, Germany) and incubated at 37 °C in an anaerobic jar for 18 h and maintained on MRS agar plates (MRSA; Merck). Bacillus subtilis (BM19) was grown in Yeast Extract Tryptone (2xYT) broth (5 μg/ml Chloramphenicol) and Bacillus subtilis (wild type) was grown in 2xYT and incubated at 30 °C in shaker incubator for 12–18 h. 2.2. Cell-free culture supernatant preparation L. acidophilus was incubated in MRS broth at 37 °C for 18 h. Cell-free supernatant (CFS) was obtained by centrifugation of bacterial culture broth at 8000 ×g for 20 min at 4 °C. The supernatant was then sterilized by filtration through a 0.2-μm syringe filter (Millipore, Bedford, MA, USA) [9].
M. Sarikhani et al. / International Journal of Biological Macromolecules 115 (2018) 762–766
2.3. Partial purification of bacteriocin After incubation of L. acidophilus for 18 h, 100 mL of cell-free supernatant (CFS) was salted out in 60% saturated ammonium sulfate at 4 °C and the precipitate collected by centrifugation at 11,000 ×g for 30 min at 4 °C. The pellet was dissolved in 1 mL of MRS broth and the suspension was dialyzed against deionized water using dialysis tubing (12,000 kDa; Sigma) in 4 °C for 48 h [10–12].
2.4. Relative molecular weight determination by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) The relative molecular weight of extracted proteins of L. acidophilus was estimated using SDS-PAGE at 120 V for 2.5 h. The gel was then stained with the Coomassie Blue R250 [14]. The molecular weight of the protein(s) was determined by comparison with the protein molecular size marker (prestained protein ladder, 11–180 kDa, CinaClon, Tehran, Iran).
2.5. The study of antibacterial properties The inhibitory effects of partially purified CFS of L. acidophilus on B. subtilis strains were studied by microscale optical density assay (MODA). Initially, test culture was added to each well of a 96-well plate; this was carried out for 5 wells for each test culture. To the first well, nothing was added (no supernatant or media); to the second, 100 μL of the CFS was added; the remaining CFS was adjusted to pH 6.0 with 10 N NaOH in order to remove possible inhibition effects due to organic acids, and then 100 μL of the pH-adjusted CFS was filtered and added to the third well. To remove the possible inhibitory effect of H2O2, the neutralized CFS was treated with 5 mg/mL catalase (2000 units/mg) (Sigma) at 25 °C for 1 h and filtered, then loaded into the fourth well. After neutralization of acid and removal of H2O2, it was treated with 1 mg/mL of proteolytic enzymes including pepsin (2500 units/mg) (sigma) and trypsin (2000 BAEE unit/mg) (Sigma) at 37 °C for 2 h and added to the fifth well. In all wells, MRS broth was applied in similar conditions to the CFS as a control. 100 μL of bacterial suspention in 2xYT (diluted until an absorbance of 0.08–0.13 at 595 nm equal to 0.5 McFarland) was then added to each well. Each series was run in duplicate on the same plate which was then incubated at 30 °C for 12–18 h. After incubation, the absorbance was read by a microplate reader at 600 nm. The difference in absorbance between the control and samples was used to report antibacterial activity [13].
2.6. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of bacteriocin against planktonic state in presence and absence of IPTG
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2.7. The effect of HBsu nucleoprotein on biofilm formation of Bacillus subtilis BM19 Bacillus subtilis BM19 cells were grown in 2xYT broth with 1 mM IPTG and 5 μg of chloramphenicol per ml at 30 °C overnight, and was diluted with fresh 2xYT medium to obtain 0.5 Mc.Farland turbidity. It was further diluted 1:100 and 200 μL of the bacterial suspension, in the presence of 0, 0.1, 1 mM IPTG and 5 μg/mL of chloramphenicol, was added to 96-well flat-bottomed polystyrene plate and incubated at 30 °C for 24 h. After biofilm formation, the medium was aspirated, and the plate washed 3 times with 200 μL of PBS to remove unbound cells after which biofilms were stained with crystal violet 1% w/v (Sigma) for 2 h. Crystal violet staining was used to measure the amount of biofilm in presence of different amounts of IPTG. The wells were rinsed and filled with 33% glacial acetic acid (v/v). After 20 min of incubation, the plates were vigorously shaken and the absorbance was read at 450 nm [17,19]. 2.8. The inhibitory effect of bacteriocin against biofilm formation in presence and absence of IPTG The Bacillus subtilis (wild type) and B. subtilis BM19 were grown in 2xYT broth medium (5 μg/mL Chloramphenicol) with 1 mM IPTG at 30 °C overnight, then diluted with fresh 2xYT medium until an absorbance of 0.08–0.1 at 595 nm was obtained equal to 0.5 McFarland. 100 μL of the bacterial suspension in the presence and absence of IPTG as well as 100 μL of the L. acidophilus - purified CFS containing bacteriocin was added to a 96-well flat-bottomed polystyrene plate and incubated at 30 °C for 24 h. The wells containing sterile 2xYT and bacterial suspension without treatment were used as blank and control, respectively. Following biofilm formation, the media was aspirated, and the plate was then washed 3 times with 200 μL of PBS to remove unbound cells. The same conditions were maintained for control wells except PBS was used instead of peptide solution as the negative control. After removal of the contents from the wells, biofilms were stained with crystal violet 1% w/v (Sigma) for 2 h. Crystal violet staining was used to measure the amount of biofilm of bacterial cells that survived the treatment with peptide solution. The wells were then rinsed and filled with 33% glacial acetic acid (v/v). After 20 min of incubation, the plates were vigorously shaken and the absorbance was read at 450 nm [17,18]. 2.9. Statistical analysis The experiments were repeated three times and to determine whether there are any statistically significant differences between the means of three or more independent groups, the one-way analysis of variance (ANOVA) and independent samples t-tests were used to analyse results of strains and the significance level was set at P b 0.05. The statistical analysis was conducted using SPSS 20. 3. Results
Minimum inhibitory concentration (MIC) values for B. subtilis BM19 and B. subtilis (wild type) were determined by the broth microdilution procedure. The MRS broth containing bacteriocin was added to microtiter plate containing same amounts of 2xYT broth and serial dilutions were prepared. The minimum bactericidal concentration (MBC) values were measured by determining the lowest concentration of antimicrobial that would prevent the growth of an organism after subculture on 2xYT agar. The B. subtilis strains, precultured in 2xYT medium at 30 °C overnight, were inoculated into fresh 2xYT broth medium and incubated until an absorbance of 0.08–0.13 at 595 nm was obtained (equal to 0.5 McFarland). Then 100 μL of the purified CFS contain L. acidophilus bacteriocin was added to the wells of 96-well plates containing 100 μL of bacterial suspensions of BM19 with 0 & 1 mM IPTG and 5 μg/mL chloramphenicol and BM19 (wild type) and then incubated at 30 °C in shaker incubator [15,16].
3.1. Partial purification of L. acidophilus bacteriocin After partial purification of CFS by salting out method and dialysis against deionized water, antibacterial protein was loaded on SDS PAGE and results suggest that there are 2 protein bands with 48 and 68 KD molecular weight (Fig. 1). 3.2. The antibacterial effect of L. acidophilus CFS on B. subtilis strains The untreated CFS of L. acidophilus showed an inhibitory effect on B. subtilis (wild type) (Fig. 2). To prove the presence of the antibacterial peptides, other antibacterial factors were removed from CFS. Organic acids were removed by CFS neutralization with 10 N NaOH. To eliminate the possible effects of H2O2, the CFS treated with catalase and all results
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Lane 2
Lane 1
Table 1 Minimum inhibitory concentrations (MICs) of antimicrobial peptides (μg/mL).
180 135 100 75 68 KDa
63 48
48 KDa
35 25
17 11 Fig. 1. SDS-PAGE of bacteriocin from L. acidophilus. Lane1: Protein molecular size marker (prestained protein ladder); Lane 2: Protein bands stained with Coomassie Blue R250 (partial purified with 60% ammonium sulfate saturated and dialysed).
compared with the controls (Fig. 2). The results indicated that antibacterial effect of CFS was not related to other factors from bacteria and moreover, organic acids and H2O2 had no effect on the antimicrobial activity of the CFS. However, treatment of CFS with proteolytic enzymes (pepsin and trypsin) decreased the antimicrobial activity of CFS of L. acidophilus on B. subtilis, so in the absence of CFS proteins, B. subtilis could grow and no antibacterial effects was observed (P b 0.05) (MRS broth used as a control) (Fig. 2). 3.3. Determination of MIC and MBC
Bacterial strains
MIC of bacteriocin (μg/mL)
MBC of bacteriocin (μg/mL)
B. subtilis (wild type) B. subtilis BM19 (1 mM IPTG) (5 μg/mL Chloramphenicol) B. subtilis BM19 (0 mM IPTG) (5 μg/mL Chloramphenicol)
0.80 0.80
0.80 0.80
0.10
0.20
planktonic cells. In the presence of IPTG, the bacteria is more resistant to bacteriocin than when IPTG is not present to induce expression of HBsu. Furthermore, bactericidal effect of bacteriocin decreases when there is IPTG in the medium to induce expression of HBsu (Table 1). 3.4. The effects of HBsu on biofilm formation of Bacillus subtilis HBsu is a nucleoprotein with different effects on bacterial cells and here our study indicates that it has a role in biofilm formation of bacteria since when there is same amounts of bacteria in wells of microtitre plate in start of incubation, in the absence of IPTG that can induce expression of HBsu, biofilm formation of Bacillus subtilis was very low and with increasing IPTG in medium, biofilm formation increased (wild type B. subtilis used as a control) (Fig. 3). 3.5. The inhibitory effect of bacteriocin against biofilm of B. subtilis strains in presence and absence of IPTG (1 mM) The inhibitory effect of bacteriocin of L. acidophilus against B. subtilis strains were determined using polystyrene microtiter plate following staining of the cells and reading the absorbance (Table 2). Bacteriocin of L. acidophilus can limit biofilm formation by B. subtilis and in the absence of IPTG which induces expression of HBsu, B. subtilis BM19 is more sensitive than when HBsu expressed in bacterial cells. 4. Discussion It has been proposed that most bacteria in nature are found in a biofilm mode of growth [20]. Biofilms are also found in medical and industerial settings, where they can be problematic due to the increased resistance of biofilm cells to antimicrobial agents [21]. A general problem caused by biofilms is that some cellular by-products accelerate corrosion of stainless steel. Other problems include biofilm formation in
The MIC of bacteriocin which was purified from the L. acidophilus CFS against B. subtilis strains was determined using a microdilution method (Table 1). Antimicrobial activity of bacteriocin produced by L. acidophilus against B. subtilis strains suggest that bacteriocin could inhibit
Fig. 2. MODA of CFS from L. acidophilus on B. subtilis (wild type) growth. Data represent the mean and standard deviation (±SD) of 3 different experiments performed in triplicate (P b 0.05).
Fig. 3. Effect of HBsu on biofilm formation of B. subtilis BM19 (5 μg/mL Chloramphenicol). Data represent the mean and standard deviation (±SD) of 3 different experiments performed in triplicate (P b 0.05).
M. Sarikhani et al. / International Journal of Biological Macromolecules 115 (2018) 762–766 Table 2 Inhibitory effect of Bacteriocin in presence and absence of IPTG (5 μg/ml Chloramphenicol) in B. subtilis. Bacterial strains
OD (Control)
OD (Treated with bacteriocin)
B. subtilis (wild type) B. subtilis BM19 (1 mM IPTG) B. subtilis BM19 (0 mM IPTG)
1.87 1.80 0.12
0.84 0.39 0.11
heat exchangers that reduce the fluid flow and the heat transfer and in membrane filtration units, biofilm block membrane pores, which decrease the flux of permeate, degrades the membranes, and reduce the trans-membrane pressure [22]. In this study, Gram positive B. subtilis was selected due to its ability to differentiate into many distinct cell types: vegetative cells, spores and biofilms [22]. Therefore B. subtilis was a suitable candidate to study the effect of HBsu nucleoprotein in biofilm formation and resistance of its planktonic form and biofilm to bacteriocin as an antimicrobial agent. Among Gram positive bacteria, the molecular mechanisms of biofilm formation appear to be specific [23]. In this study, for the first time, we studied the effect of HBsu nucleoprotein on biofilm formation of B. subtilis and antibacterial effects of Lactobacillus acidophilus bacteriocin on planktonic cells and biofilm formation of Bacillus subtili was studied. HBsu is an important nucleoprotein in Bacillus subtilis and plays different roles in bacterial cells. Initially, bacteriocin was purified by salting out method and to detect the molecular weight of this protein, it was loaded on SDS-PAGE gel. There were two proteinaceous bands with 48KDa and 68 KDa weight which were relatively large and belonged to class III (N30 kDa) bacteriocins [24]. To detect antimicrobial effect of bacteriocins on B. subtilis, purified CFS from L. acidophilus containing bacteriocin was added to the media containing B. subtilis. To determine whether this antimicrobial effect is related to bacteriocin, the bacteriocin-like substances were removed with MODA and therefore it seems bacteriocin of L. acidophilus has an inhibitory effect on growth of B. subtilis. Vahedi Shahandashti et al. (2016) studied the effect of this bacteriocin on Gram negative bacteria, Serratia marcescens, and showed that it had inhibitory effects on growth and biofilm of this bacteria and the MIC of bacteriocin against S. marcescens ATCC 13880 and ATCC 19180, was respectively N0.12 and 0.12. Salman et al. (2014) studied antibacterial and antibiofilm effect of L. acidophilus on Gram positive bacteria, Bacillus cereus, Staphylococcus aureus and Bacillus subtilis and showed that bacteriocin displayed bacterial growth inhibition as 72.79%, 87.12% and 96.42%, respectively and their microscopic studies showed that there was an increased biofilm inhibition activity in highly purified bacteriocin extract [26]. Vilela et al. (2015) surveyed the effects of L. acidophilus ATCC 4356 on biofilm formation by C. albicans and the highest inhibition (57.52%) was observed after 24 h of L. acidophilus culture. Furthermore, they showed that L. acidophilus culture filtrate reduced the growth of C. albicans cells by 45.10%, suggesting that L. acidophilus produced substances with anti-Candida activity [27]. Campana et al (2012) showed that the co-culture of L. acidophilus ATCC 4356 was able to inhibit the growth of C. jejuni. In most cases, the growth reduction of C. jejuni strains was obtained by 6, 9, and 24 h of co-culture, with the highest values of growth inhibition of 27.31, 25.10, and 26.94% for C. jejuni Hom 13, C. jejuni ISS 3, and C. jejuni ISS 9, respectively. Moreover, the CFS of L. acidophilus ATCC 4356 at pH 6.5 showed inhibitory activity against eight of the human C. jejuni strains and their findings considered that the inhibitory action could be due to a proteinaceous molecule [28]. Goudarzi et al. (2014) studied antimicrobial activity of bacteriocin produced by Lactobacillus bacteria against Proteus species and their results demonstrated that the cell-free supernatant from L. plantarum and L. acidophilus ATCC 4356 is effective in inhibiting the growth of different strains of Proteus [29]. In this research, the partially purified bacteriocins of L. acidophilus were added to B. subtilis media and MIC of bacteriocin on B. subtilis (wild type), BM19 in present and absence of IPTG (inducer of HBsu)
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were respectively 0.80, 0.80,0.10 (μg/mL). The results showed that in the absence of HBsu nucleoprotein, bacteria are more sensitive to bacteriocin and compared with Vahedi Shahandashti (2016), MIC and MBC of B. subtilis were more than S. marcessence therefore it can be postulated that in Gram positive bacteria, a thicker peptidoglycan be the cause [12]. Brigit M et al. (1991) showed the dependence of bacterial growth on HBsu. Here, for the first time, we report the effects of HBsu on biofilm formation of B. subtilis BM19. Identical amounts of bacteria (0.5 McFarland) were loaded to wells of microtitre plate with one containing 1 mM IPTG and 5 μg/mL chloramphenicol and in other another 0.1 mM IPTG and 5 μg/mL chloramphenicol. After following incubation, results showed that in the presence of HBsu nucleoprotein, biofilm formation is completed but is aborted when HBsu is not sufficient in the bacterial cells. Biofilm formation of B. subtilis was also limited by bacteriocin of L. acidophilus [5]. Lipsy Chopra (2015) studied the effect of bacteriocin on another Gram positive bacterium and showed that bacteriocin can inhibit attachment of biofilm to surface [25]. In the absence of IPTG, biofilm formation was less than the time there was IPTG in media. We suggest that HBsu nucleoprotein is an important agent in biofilm formation of B. subtilis BM19 and is effective on the resistance of planktonic cells and biofilms of B. subtilis BM19 to L. acidophilus ATCC4356 bacteriocin. which can limit biofilm formation in B. subtilis.
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