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ЭЛЕКТРОННЫЙ НАУЧНЫЙ ЖУРНАЛ «APRIORI. CЕРИЯ: ЕСТЕСТВЕННЫЕ И ТЕХНИЧЕСКИЕ НАУКИ»
№ 5 2014
УДК 759.873.088.5:661.185
THE EFFECT OF ACINETOBACTER CALCOACETICUS IMV B-7241 SURFACTANTS ON MICROBIAL ADHESION TO ABIOTIC SURFACES
Savenko Inga Vladimirovna
student
Pirog Tetyana Pavlivna
doctor of biological sciences
Skrotska Oksana Igorivna
candidate of biological sciences National university of food technologies, Kyiv (Ukraine)
author@apriori-journal. ru
Abstract. We have studied the effect of surface-active substances (SAS, surfactants) of Acinetobacter calcoaceticus IMV B-7241 with various degree of purification (the supernatant of culture liquid, the solution of SAS, 0,0010,036 mg/ml) for the attachment of bacteria and fungi to abiotic surfaces. The degree of adhesion of the test-cultures depended on the material's type and the concentration of SAS in the preparations. The preparation 1 (supernatant) with the concentration of SAS 0,005-0,009 mg/ml was the more effective: after treatment of the abiotic materials with this preparation the number of attached cells of bacteria and fungi decreased on average 45-60 %.
Keywords: Acinetobacter calcoaceticus IMV B-7241; adhesion; surfactants; abiotic materials.
ВЛИЯНИЕ ПОВЕРХНОСТНО-АКТИВНЫХ ВЕЩЕСТВ АСШЕТОБАСТЕП САЬСОАСЕТЮиЭ ИМВ В-7241 НА АДГЕЗИЮ МИКРООРГАНИЗМОВ К АБИОТИЧЕСКИМ ПОВЕРХНОСТЯМ
Савенко Инга Владимировна
студент
Пирог Татьяна Павловна
д-р биол. наук
Скроцкая Оксана Игоревна
канд. биол. наук
Национальный университет пищевых технологий, Киев (Украина)
Аннотация. Исследовано влияние поверхностно-активных веществ (ПАВ) Acinetobacter calcoaceticus ИMB B-7241 различной степени очистки (супернатант культуральной жидкости, раствор ПАВ, 0,001-0,036 мг/мл) на прикрепление некоторых бактерий и грибов к абиотическим поверхностям. Степень адгезии тест-культур зависела от типа материала и концентрации ПАВ в препаратах. Более эффективным оказался препарат 1 (супернатант) с концентрацией ПАВ 0,005-0,009 мг/мл, после обработки которым наблюдали уменьшение количества прикрепленных клеток бактерий и грибов в среднем на 45-60 %.
Ключевые слова: Acinetobacter calcoaceticus ИMB B-7241; адгезия; поверхностно-активные вещества; абиотические материалы.
Introduction. A wide range of materials is presently used in various branches of industry, such as Dutch tile, steel, plastic, and linoleum (polyvinylchloride). Biofilm formation by microorganisms contaminating food staples, revetment and coating of production areas, and packaging materials in the food industry is a considerable problem [1]. It is known that most synthetic disinfectants do not penetrate deep into the biofilm; hence, disin-?ection is only partial (only the upper biofilm layer is destroyed. Microbial surfactants change the surface charge and, consequently, the cells do not adhere to the material treated with these agents [2].
Previously, we isolated oiloxidizing bacteria from oilcontaminated soil samples, which were identified as Acinetobacter calcoaceticus K-4 [3]. This strain registered at the Depository of Microorganisms of the Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, under accession numbers IМВ В-7241.
The ability of A. calcoaceticus IMV B-7241 to synthesize the low molecular weight surface-active substances on the hydrophobic and hydrophilic substrates was established. It was shown that surfactants of IMV B-7241 strain are complex of glyco-, amino and neutral lipids [3].
Previously [4] it was found that the SAS of A. calcoaceticus IMV B-7241 decreased the number of the attached to medical materials cells of certain bacteria and fungi.
The purpose of this work is to investigate the effect of A. calcoaceticus IMV B-7241 surfactants on the microbial adhesion to the abiotic surfaces.
Research methods. A. calcoaceticus IMV B-7241 was grown in liquid mineral medium containing the following (g/l): NaCl - 1,0; Na2HPO4 - 0,6; (NH2)2CO - 0,35; KH2PO4 - 0,14; MgSO47H2O - 0,1; рН 6,8-7,0. The medium was additionally supplemented with yeast autolysate, 0.5% (vol/vol), and solution of trace elements, 0,1 % (vol/vol). Ethanol at a concentration of 2 % (vol/vol) was used as a carbon source. Culture in the exponential growth
phase cultivated in the respective liquid media containing 1% (vol/vol) of the
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substrate were used as inoculum. The amount of inoculum (104-105 cells/mL) was 5-10 % by volume of the nutrient medium. The bacteria were cultivated in 750 mL flasks with 100 mL of the medium on a shaker (320 rpm) at 28-30 °C for 120 h.
These preparation of surfactants were used in studies:
Preparation 1 - supernatant of culture liquid, to obtain which the culture broth was centrifuged (5000 g, 45 min). The surfactant-containing supernatant was subjected to extraction with the 2:1 chloroform/methanol (Folch) mixture to isolate the surfactant (preparation 2). Extracts were evaporated using an IR-1M2 rotory evaporator (Russia) at 60 °C and absolute pressure of 0,4 atm to a constant mass.
The dry remnant was diluted in sterile phosphate buffer (0,1 M; pH 7,0). The preparations 1 and 2 were sterilized at 112 °C, 30 min.
The SAS concentration in preparations 1 and 2 was established by the weight technique after extraction with a Folch mixture [3].
The strains of bacteria (Bacillus subtilis ET-2, Escherichia coli IEM-1) and fungi (Candida albicans fl-6, Aspergillus niger P-3, Fusarium culmorum T-7) used in this work were obtained from the collection of the living microorganisms of the department of biotechnology and microbiology, National university of food technologies.
Antiadhesive properties were investigated as follows: the purified plates of materials (Dutch tile, stainless steel, plastic, polyvinylchloride) of the same size (1 cm2) were sterilized at 112 °C for 40 min and then were immersed into the solution of preparations 1-2 and dried for 24 h in a thermostat at 30 °C. One-day bacterial and yeast test cultures and three-day micromycete cultures grown on meat peptone agar (MPA) and glucose-potato agar (GPA), respectively, were suspended in 100 mL of sterile tap water; the materials pretreated with preparations 1-2 and untreated (control) samples were placed into the suspension, incubated for 2 h in a thermostat at 30 °C, and rinsed with 10 mL
of sterile tap water to remove non-adherent cells [5]. Then the degree of cell adhesion was determined by two methods.
Spectrophotometry method [6; 7]. The plates of materials were treated with methanol (99 %) for 15 min to fix the attached cells, dried at room temperature, placed for 5 min into 1 % gentian violet solution, and rinsed with tap water. After drying, the materials were treated with 10 mL of 33 % acetic acid solution and the optical density of the resultant suspension of desorbed cells was measured. The number (%) of attached cells (adhesion) was determined as a ratio of the optical density of the suspension obtained from the samples treated with preparations 1-2 to the optical density of the control samples (100 %).
Koch's method [8]. The materials were placed into flasks with 20 mL of sterile tap water and glass beads and shaken for 5 min to desorb the attached cells. The number of living cells in the resultant suspension was measured by the Koch's method on MPA (for bacteria) and GPA (for yeasts and micromycetes). The number (%) of attached cells (adhesion) was determined as a ratio of cells on the samples pretreated with preparations 1-2 to the cells on the control samples (100 %).
All experiments were performed in triplicate. Statistical analysis of experimental data was performed by Lakin [9]. The differences between the average values were considered reliable at a confidence level p < 0,05.
Results and discussion. Adhesion of microorganisms to certain surfaces is known to depend on the nature of their surface structures and the properties of the material [10]. In the work [11] fraction-1 of the lipopeptide surfactants from B. licheniformis V9T14 at a concentration of 0,08 mg/mL was shown to inhibit the adhesion of E. coli CFT073 to polystyrene plates by 50 %, while fraction-2 (at the same concentration) inhibited it by 90-95 %. A 100 % decrease in adhesion of E. coli CFT073 cells was obsreved in the presence of two fractions of lipopeptides from B. subtilis V19T21 (35 ^g/mL) [11].
At the first stage our research was directed on determining antiadhesive properties of preparations 1 and 2 (0,036-0,001 mg/mL) for B. subtilis vegetative cells ET-2 (14 h growth) (Table 1). The results presented in Table 1 show that decline of SAS concentration in preparations 1 and 2 was accompanied by decreasing the adhesion of ET-2 strain cells.
Our studies (Table 1) revealed that preparation 1 (0,005 mg/mL) was a more effective antiadhesive agent than preparation 2: after the treatment of surfaces with supernatant adhesion of vegetative cells of B. subtilis ET-2 on plastic, polyvinylchloride, Dutch tile and steel was 15, 20, 14 and 12 %, respectively. Further experiments showed that the surfactant preparations from A. calcoaceticus 1MB B-7241 decreased the adhesion of B. subtilis BT-2 spores to the tested materials, and the degree of adhesion actually did not differ from that for the vegetative cells.
Table 1
The adhesion vegetative cells of B. subtilis ET-2 to abiotic surfaces after the treatment with surfactants of A. calcoacetius IMV B-7241
Preparation Concentration, mg/mL Adhesio n, %
Plastic Polyvinylchloride Dutch tile Steel
1 (supernatant) 0,036 62 78 76 75
0,018 57 63 42 70
0,009 38 45 32 39
0,005 15 20 14 12
0,003 28 35 23 24
0,001 33 38 42 39
2 (surfactant solution) 0,036 72 68 59 69
0,018 46 53 52 49
0,009 41 48 48 45
0,005 23 35 35 33
0,003 31 40 41 37
0,001 38 45 51 44
Note. Tables 1-3: the adhesion error was no more than 5 %.
On the next step the effect of preparation 1 and 2 from A. calcoaceticus IMV B-7241 on adhesion cells of E. coli IEM-1 to the abiotic materials were investigated (Table 2). Both preparations at low concentrations showed an antiadhesive effect. Preparation 2 (surfactant solution, 0,005 mg/mL) was a more effective antiadhesive agent than preparation 1: the number of attached cells of E. coli IEM-1 to abiotic surfaces treated with preparation 2 decreased by 65-70 %.
Table 2
The effect of surfactant preparations from A. calcoaceticus IMV B-7241 on attachment of E. coli IEM-1 cells to abiotic materials
Preparation Concentration, mg/mL Adhesio n, %
Plastic Polyvinylchloride Dutch tile Steel
1 (supernatant) 0,036 79 70 86 92
0,018 69 58 81 72
0,009 66 55 72 65
0,005 52 42 48 48
0,003 86 58 68 88
0,001 90 88 77 92
2 (surfactant solution) 0,036 66 61 84 79
0,018 55 52 48 73
0,009 52 48 43 69
0,005 28 30 26 36
0,003 59 79 52 56
0,001 69 82 61 70
Table 3 shows the data on adhesion of C. albicans D-6 cells to abiotic surfaces treated with preparations 1 and 2 from the strains 1MB B-7241. After treatment of studied materials with supernatant and surfactant solution (0,005 mg/mL) adhesion of C. albicans D-6 cells was 30-35 %.
Investigation of the effects of preparations 1 and 2 from A. calcoaceticus 1MB B-7241 at a concentration of 0.009 mg/mL on attachment of the micro-mycete A. niger P-3 cells to plastic, polyvinylchloride, Dutch tile, and steel
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showed that they had nearly the same antiadhesive effects: adhesion to all materials was 52-62 % (Figure 1).
Table 3
The adhesion of C. albicans cells fl-6 to different materials after the treatment with surfactants of A. calcoacetius IMV B-7241
Preparation Concentration, mg/mL Adhesio n, %
Plastic Polyvinylchloride Dutch tile Steel
1 (supernatant) 0,036 74 70 61 73
0,018 65 57 51 55
0,009 39 39 44 49
0,005 31 28 36 35
0,003 41 45 51 55
0,001 72 76 78 75
2 (surfactant solution) 0,036 67 55 49 60
0,018 61 51 46 51
0,009 33 36 40 42
0,005 28 25 30 32
0,003 37 39 43 45
0,001 87 88 85 88
70 60 50 40 30 20 10 0
Plastic
Polyvinylchloride
Dutch tile Steel
Figure 1. The effect of A. calcoaceticus IMV B-7241 surfactants (0,009 mg/mL) on adhesion of cells of A. niger P-3 to abiotic surfaces
A similar patterns were also observed for the cells of F. culmorum T-7: the degree of cell adhesion was 60-65 % after the treatment of tested materials with preparations 1 and 2 from A. calcoaceticus 1MB B-7241.
Conclusions. Thus, the surfactant preparations from A. calcoaceticus 1MB B-7241 with different degrees of purification (both as supernatant of culture liquid and as a solution of extracted surfactants) can be used for development of highly efficient preparations, decreasing microbial adhesion to the surface of different materials. Note that it would be more economically expedient to use preparation 1 (supernatant) because the technology of its production presupposes no additional stages of isolation and purification.
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