The role of probiotic Lactobacillus acidophilus ATCC 4356 bacteriocin on effect of HBsu on planktonic cells and biofilm formation of Bacillus subtilis

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Abstract

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.

Introduction

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.

Section snippets

Bacterial strains and culture conditions

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

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).

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

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 heat exchangers that reduce the fluid flow and the heat transfer and in

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