Zh Mikrobiol Epidemiol Immunobiol. 2012.

4:66-70 [Article in Russian]

Influence of antimicrobial peptides of human platelets on Staphylococcus

aureus biofilm formation
Zhurlov O.S.1, Perunova N.B.1,3, Ivanova E.V.1,3, Egorova O.S.2
1

Institute of Cellular and Intracellular Symbiosis, UB RAS, Orenburg, Russia

Institute of Macromolecular Compounds, Russian Academy of Sciences, St. Petersburg, Russia
3
Medical University Orenburg State Medical Academy, Orenburg, Russia
2

Abstract
AIM:
Study the influence of platelet low molecular weight protein on S. aureus biofilm
formation.
Materials and methods:
20 clinical isolates of coagulase-positive staphylococci (S. aureus) served as material.
Preparation containing a mixture of antimicrobial peptides (fractions with molecular weight 6070 kDa, 20-25 kDa and 5-10 kDa) obtained from human thrombocyte donors was used. Biofilm
formation (BF) by staphylococci was studied photometrically, the degree of hydrophobicity was
evaluated by separation of cell suspension in two-phase system liquid-liquid.
The data obtained were treated by nonparametric method using Mann Whitney criteria.
Results:
Platelet low molecular weight proteins (PLMWP) at concentration of 2 mg/ml reduced
biofilm formation of S. aureus by 48.79.8 % from initial values, increased hydrophobicity of
plankton and biofilm cell fractions. Maximum inhibitory effect of the preparation containing
platelet low molecular weight proteins was noted at concentration of 50 mg/ml.
Conclusion:
The data obtained on inhibition of staphylococci BF by platelet low molecular weight
protein open a perspective of further studies of PLMWP as a preparation suitable for control of
biofilm of persistent microorganisms.
Key words: platelet low molecular weight protein (PLMWP), biofilm, hydrophobicity
Introduction
Bactericidal factors of cells and tissues perform a crucial function in the protection of
microorganism, especially in the initial phases of infection [3]. Among them a special place is
occupied by low molecular weight proteins with severe cationic properties, which include
platelet cationic proteins (PCPs). Known membrane platelet activity of the protein, the ability to
inhibit enzymes of antioxidant protection - peroxidase and catalase, cause accumulation of lipid

peroxidation products, with which it is associated with the bactericidal action. Also found to
reduce bacterial adhesion PCPs at somatic cells, and changes the characteristics of the
persistence Escherichia coli and Staphylococcus aureus [4]. Previously conducted studies have
shown that the biological activity of the PCPs is not limited to its bactericidal function, it shows
the involvement of the peptide in a wide range of protective and adaptive reactions, reflecting the
state of immune homeostasis and general [2]. The possible universality of the biological effects
of various cationic proteins in infectious pathology, and homeostasis.
In the last decade the formation of antibiotic resistant forms of microorganisms necessitates
the search for new means of treatment of infectious diseases. This is especially true against
staphylococci able to form biofilms and long-term survival in tissues and organs, demonstrating
a wide range of adaptive capabilities [5].
The ability of bacteria to adhere on abiotic surfaces and as the initial stage of biofilm
formation, often has non specific bacteria and is realized by a modification of its own bacterial
surface [14], i.e. its physicochemical properties (hydrophobicity and zeta potential). In recent
years began to appear about the impact of natural antimicrobial compounds on hydrophobic and
donor-acceptor properties of the surface of microbial cells that are important in the formation of
bacterial sensitivity to the drug [8].
However, in the literature there are no data on the effects of low-molecular platelet proteins
on the physicochemical properties of Staphylococcus aureus and their ability to biofilm
formation.
Therefore, the aim of the study was to investigate the effect of platelet derived low
molecular weight protein (LMWP) on the physicochemical properties of S.aureus and their
ability to biofilm formation.

Materials and methods

Research material 20 clinical isolates S. aureus (from collection of ICVS UB RAS).
Identification of microorganisms was carried out on the basis of generally accepted methods of
morphological, tinctorial, cultural and biochemical properties. To identify the system used
standard StaphyTest (LaChema, Czech Republic).
We used a lyophilized preparation containing the mixture of antimicrobial peptides from
human platelets (PLWP). The formulation was prepared from the platelet-containing platelet
0.55 1011. Subjected to 3x freeze (-110 C) and thawing (+ 3 C - + 5 C) and centrifuged at
1000g for 30 minutes. The supernatant was filtered through membrane filters Durapore 0.22
microns and lyophilized.

PAGE electrophoresis in the presence of SDS-Na showed that PLWP complex preparation
contains proteins with molecular weights in the 60 - 70 kDa protein with a mass of 20-25 kDa
and low molecular weight peptides with 5 - 10 kDa. Protein content was determined by the
Bradford method [9], which was 38%.
Drug activity PLWP relative to the test strains of gram-positive bacteria M. lysodeikticus
2665 and S. aureus P 209 determined plate method [6]. MIC50 to M. lysodeikticus 2665 was 5
mg/ml, and for S. aureus P 209 - 1 mg/ml.
Biofilm formation (BF) of bacteria was studied using a photometric method [12], by
growing S.aureus in broth 150 mkl in a 96-well polystyrene (hydrophilic) plate for 24 hours at
37C, removal from the wells of planktonic cells staining received 1% biofilm with crystal violet
solution, washing the wells with distilled water to make 96% of ethanol and acetic acid and
measuring the optical density (OD) with a photometer Multiscan ascent (Thermo Electron
Co., China) at a wavelength ( ) 570 nm. The intensity of staining consistent with the degree of
biofilm formation studied cultures of Staphylococcus aureus. For conventional units (OD)
biofilm formation activity of microorganisms took the ratio of the optical density in the
experiment (OD broth culture of microorganisms) and control (OD nutrient broth).
Planktonic and biofilm cells were selected as follows: after 24 hours of growth, 96-well
polystyrene plates were taken on a 100 mkl suspension in broth planktonic cells. Removed the
remains of the broth and planktonic cells of the microtiter plate. Was added to the wells 100 mkl
of 0.9% NaCl solution and disintegrate the biofilm collected biofilm 100 mkl disintegrated cells.
Biofilm and planktonic cells, 3-fold, washed from the broth at 250g. Suspensions were
standardized in NF at () of 540 nm, and subsequently used to measure the hydrophilic-lipophilic
balance (HLB).
To evaluate the degree of hydrophobicity separation method using staphylococcus cell
suspension in a biphasic system of "liquid-liquid" aqueous phases with 0.9% solution of NaCl,
enriched polyethylene glycol (PEG 6000, final concentration 4.5%) and dextran (T500 c final
concentration 6.2%) [1].
Measurement of zeta potential (zeta potential, mV) cells staphylococci carried amplitudefrequency method using "Dzetometr-1M" in the normal mode of his work (voltage - 10 V, the
frequency - 0.2 Hz) by measuring the amplitude of the oscillations 50 bacterial cells
microelectrophoresis chamber (2222 mm, height 0.2 mm), and calculating average values for
the zeta potential on the approximated strain Smoluchowski formula [13].

Statistical analysis

Data processing was carried out by methods of variation statistics and correlation analysis
[7] and nonparametric method using Mann Whitney criteria.
Results
It was revealed that the platelet containing complex preparation of low molecular weight
proteins having a low antibacterial activity against the test strains of staphylococci (PLMWP
only at concentrations of 100 mg/ml), but decreased its ability to form a biofilm.
The minimum concentration, at which the inhibition of biofilm formation observed cultures
S.aureus, was 2 mg / ml. At the same time, the average values of BPO staphylococci declined to
48.7 9.8% from baseline values. Indicators S.aureus biofilm formation in the control (without
drug exposure) were 1.99 0.01 OD570, whereas after the impact of PLMWP in a concentration
of 2 mg/ml of the figure dropped to 1.09 0.01 OD570, (p <0.05 ). Maximal inhibitory effect of
the drug-containing low-molecular platelet-derived peptides was observed at a concentration of
50 mg/ml and produced an average reduction in biofilm formation values S.aureus at 87.4
6.5% of baseline values. The average values of BF staphylococci in control were 1.99 0.01
OD570 (p <0.05).
To explain the phenomenon of reduction in biofilm formation at the 8 S.aureus strains
PLMWP interacting with a concentration of 2 mg/ml conducted definition of hydrophobicity
(HLB cells selected from the planktonic and biofilm fraction). Hydrophobicity native biofilm
formation S.aureus averaged (-0.85 0.1 optical units), plankton (-0.63 0.2 optical units) and
biofilm cells (-0.79 0.1 optical units).
Incubation with staphylococcal preparation in PLMWP working concentration of 2 mg/ml
resulted in an increase in hydrophobicity as planktonic (-0.45 0.1 optical units), and biofilm
fraction of cells staphylococci (-0.55 0.1 optical units). To confirm increasing hydrophobicity
cells associated with interaction with PLMWP rather than mechanical disintegration of biofilm
cells, a series experiments was conducted using the same strains of staphylococci. The
experimental results showed that the mechanical disintegration of the biofilm cells did not alter
the degree of hydrophobicity.
Discussion
At present, the role microbial biofilms proved in the development a number human
infections [10]. A distinctive feature of the bacteria in the biofilm community composition, is to
increase by 1-2 orders of antimicrobial resistance [11], which creates significant difficulties for
antimicrobial therapy. Unfortunately, standard methods of antibiotic treatment aimed at existing
separately planktonic cells, whereas bacteria multiply inside the biofilm of disseminated and
again after completion of treatment, often forming chronic persistent infection, contributing to

recurrence to the disease [11]. In this connection, it is necessary to evaluate not only the effect of
antimicrobial agents, but their effect on microbial biofilm formation.
Submitted in evidence not only of the presence of bactericidal activity against grampositive bacteria PLMWP, as shown earlier, but the suppression of biofilm formation in clinical
isolates of S.aureus, is part of the agents of inflammatory diseases in hospitals in various fields.
Found that the minimum concentration of drug PLMWP inhibiting biofilm formation
staphylococci is 2 mg/ml, and the most effective dose PLMWP suppressing biofilm formation
S.aureus - 50 mg/ml.
Increasing HLB planktonic and biofilm fraction of cells interacting with PLMWP
preparation, indicating a change in the physicochemical properties BF staphylococci and helps
reduce bacterial adhesion to the hydrophilic surface, which reduces the bacterial biofilm
formation.
The resulting materials hold promise for further study of the drug complex comprising
platelet low molecular weight peptides, as not only an antimicrobial agent, but also as drugs that
inhibit microbial biofilm formation.

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