The antibacterial activity of probiotic peptide against of methicillin-resistant staphylococcus aureus (mrsa)

Abbas Tanhaeian,1,* Atefeh asadi,2

1. 1- Ph.D. Biotechnology, Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi university of M
2. M.sc of food science and technology, faculty of agriculture , ferdowsi university of Mashhad

Abstract


Introduction

Staphylococcus aureus is one of the most important causes of hospital infections. it has become one of the major public health concerns due to inherent ability and resistance to antimicrobial agents. this bacterium resists many antibiotics, especially beta-lactams. infections caused by this bacterium can even cause the death of patients with it. therefore, the increase in antibiotic resistance is a concern and should be controlled. the spread of staphylococcus aureus (mrsa) has been implicated in different countries. in the past, many mrsa-infections were limited to elderly people in health centers and they were described as mrsa-related health care .however, epidemiology of mrsa was admitted to the hospital with the addition of strains in patients without a history of admission, and mrsa was associated with it. this is because the strains of staphylococcus known as mrsa do not respond well to many common antibiotics used to kill bacteria. antimicrobial peptides (amps) have broad activities against gram- positive bacteria, gram-negative bacteria, fungi, viruses, parasites, even tumors and have been recognized as effector molecules in the innate immune system. some antimicrobial peptides (amps) have shown excellent effects on these drug-resistant pathogens in vitro. the amps display narrow- or broad-spectrum activity against bacteria, fungi, viruses and/or parasites including mrsa. due to their unique mode of action, antimicrobial peptides are novel alternatives to traditional antibiotics for tackling the issue of bacterial multidrug resistance. therefore, the development of new classes of amps represent promising foundations for the development of a new generation of antimicrobial agents. the use of different combinations of amps and antibiotics is a promising method to improve the activities against mrsa. antimicrobial research is being pressured to look for more effective therapeutics for the ever-growing antibiotic-resistant infections, and antimicrobial peptides (amp) and antimicrobial combinations are promising solutions. amp are good candidates due to their alternative mechanisms of action, usually involving cell membrane damage, nucleic acid, protein biosynthesis and general unspecific molecular targets, which reduces the chance of acquired resistance. antimicrobial peptides have different sources, one of the most useful of which are probiotics, including probiotic enterococcus faecium p13.this probiotic produces small amounts of bacteriocin, which has good antibacterial properties. therefore, the synthesis of this peptide in a recombinant and secretive manner in order to access appropriate quantities and easy purification of the targets of many researchers in the world. design of shorter amp sequences without toxicity and intense activity is needed to overcome to antibiotic resistance problems and produce effective therapeutic agents. here we investigated the purification, characterization and antibacterial effects of class ii bacteriocins, enterocin.

Methods

Codon optimization of enterocin dna encoding sequence for cho cell expression system was done by -tasser online server (http://zhanglab.ccmb.med.umich.edu/i-tasser/) and chemically synthesized in pgh vector (macrogen, korea). escherichia coli dh5α competent cell preparation and transformation steps were followed as described by sambrook and russell (sambrook & russell, 2001). plasmid containing inserted target dna was extracted using the plasmid dna isolation kit (fermentas, usa) and confirmed by restriction enzyme digestion. recombinant pcdna3.1+ vector containing target gen was transmitted into cho cells, using calcium phosphate method, in dmem/f12 medium which supplemented with 10% fbs and 1% pen/strep according to jordan et al. antibiotic therapy using g418 was used to verify transfection. culture medium (dmem) upon transmitted cells was collected (secretion signal peptide causes secretion of the recombinant protein into the medium). the protein was then evaluated by sds-page electrophoresis. mic test of each treatments is the absence of visual growth of bacteria, in order to determination of mic by microtiter plates and mbc by microtiter plates has been done using following the mics results.

Results

Enterocin exhibited an activity against methicillin-resistant staphylococcus aureus with the highest activity of minimum inhibitory concentration values. the mics of enterocin peptide against methicillin-resistant staphylococcus aureus after determination of their antibiotic resistance. the mics of enterocin peptide against methicillin-resistant staphylococcus aureus was 18.6 μg/ml.

Conclusion

The potent antimicrobial activities of enterocin present an attractive therapeutic candidate for treatment of methicillin-resistant staphylococcus aureus infections.

Keywords

Methicillin-resistant staphylococcus aureus; probiotic; antimicrobial peptides