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DOI: http://dx.doi.org/10.20534/ESR-17-1.2-8-10
Zhurlov Oleg Sergeevich, Institute of Cellular and Intracellular Symbiosis, Ural Branch of the Russian Academy of Sciences, Orenburg, Russia E-mail: jurlov1968@mail.ru
Comparative analysis of conversion of physicochemical properties of biofilm-forming bacteria resistant to the innate immunity factors
Abstract: We conducted a comparative analysis of conversion physicochemical properties of biofilm-forming bacteria that are resistant to the bactericidal activity of blood serum, lysozyme and fraction of low molecular weight platelet peptides. It was established that bacterial resistance to blood serum, lysozyme and low molecular weight platelet peptides correlates with low hydrophilic and high values of electrokinetic potential cells. The conversion of physicochemical properties of bacteria, during incubation with the innate immunity factors, has unidirectional character and contributes to the increase of hydrophilic cells. The shift of physicochemical properties a bacteria increases in the range - E.coli < S.aureus < coagulase-negative staphylococci.
Keywords: Escherichia coli, Staphylococcus aureus, coagulase-negative staphylococci, HLB (hydrophilic-lipophilic balance), electrokinetic potential.
Introduction
Relevance of the study a bacterial resistance to the innate immunity factors is caused by a significant growth of bacterial resistance to antibiotics [10], as well by increase in the number of immunocompromised patients [2]. Therefore, one of the important research directions is deciphering the mechanisms related to the formation of unsusceptible bacterial phenotype (morphotype), which is resistant to the innate immunity factors and antibiotics.
The combined selective effect of antibiotic therapy and innate immunity factors contributes to the selection of unsusceptible bacterial phenotype resistant to antibiotics and innate immune factors, what sometimes leads to bacteraemia and sepsis [7; 8]. The common pattern for clinical isolates of microorganisms, which were extracted from niduses of purulent-inflammatory diseases of soft tissues, is a high degree of hydrophilic of the surface both for bacterial cells [3] and yeast-like fungi of the genus Malassezia isolated from the cases of mycoses, accompanied by inflammatory changes to skin [1]. In this context, mechanisms related to the formation of resistance biofilm-forming bacteria to bactericidal activity of blood serum and low molecular weight platelet peptides represent the unquestionable interest [12; 13]. As we have previously shown [3], the main physicochemical indicator, which demonstrates the formation of resistant phenotype of microorganisms, is seen to be conversion (shift) of physicochemical properties of bacteria. Nevertheless, direction and value of conversion physicochemical properties (HLB and electrokinetic potential) of biofilm-forming bacteria resistant to the innate immunity factors remain unknown.
The objective of the study was to conduct a comparative analysis of conversion physicochemical properties of clinical isolates biofilm-forming bacteria resistant to the innate immunity factors.
Materials and methods
The research was based on biofilm-forming strains of clinical isolates of bacteria extracted from urogenital infections and from the bile of patients with an infectious-inflammatory pathology of the biliary tract (cholangitis, cholecystitis). For the study we selected biofilm-forming strains ofbacteria (E.coli (n=36), S.aureus (n=12)) and coagulase-negative staphylococci (S.epidermidis, n=6; S.hominis, n=12; S.haemolyticus, n=11). Identification of bacteria was carried out on the basis standard methods by morphological, tinctorial, cultural and biochemical properties with the use of standard systems «StaphyTest» and «EnteroTest I, II» (LaChema, Czech Republic).
In this study we used the frozen-dried preparation containing the mixture of antimicrobial peptides from human platelets (hPL), obtained from the platelet concentrates containing 0.55x10 11 platelets. Electrophoresis in polyacrylamide gel with sodium dodecyl sulfate showed that the complex product of platelet lysate (hPL) contained proteins with a molecular weight 60-70 kDa, proteins with weight 20-25 kDa and low molecular peptides with weight 7.61-10.47 kDa [4; 9]. During the experiment we used the hen egg-white lysozyme (Sigma Aldrich, USA) solution with concentration 50 ^g/ml in sterile 0.15 M NaCl, and native blood serum sampled from 20 donors (with working concentration (50%) in 0.15 M NaCl).
Comparative analysis of conversion of physicochemical properties of biofilm-forming bacteria resistant to the innate immunity factors
Biofilm formation was studied on the basis of photometric method. For gram-positive staphylococci we applied the method Knobloch J. K. et al. [5] and for biofilm-forming strain of E.coli — the method proposed by O'Toole G. F. et al. [11]. Intensity of biofilm staining was measured with a photometer «Multiscan Ascent» (Thermo Electron Co., China) at 1=570 nm. Intensity of staining corresponded to the degree of biofilm formation in the examined bacterial strains.
Bacteria resistant to the innate immunity factors (blood serum, lysozyme, antimicrobial platelet peptides) were selected as follows — after incubation of bacterial suspension (0.1 as per MFarland) with lysozyme (final concentration 25 ^g/ml), blood serum (final concentration 25%) and product of platelet lysate (20 mg/ml) during 30 minutes at 37°. Inoculum was sown in solid culture medium. On the following day CFU was estimated, all cells were harvested (suspensions were standardized) with optical density 0D540=0.2 in spectrophotometer.
In order to estimate the degree of bacterial hydrophobicity, we used the method of separation of cell suspension in two-phase system "liquid-liquid" with immiscible water phases in 0.15 M sodium chloride solution, enriched with polyethylene glycol (PEG 6000; with concentration 4.5%) and dextran (T500; with concentration 6.2%) [3]. For standardization of results, they were expressed in absorbance units (AU). Absorbance unit is equal to decimal logarithm of ratio between optical densities of upper and lower phases containing bacteria during separation of suspension into two phases, with volume of 1 ml each, measured by spectrophotometer, at 1=540 nm.
Measuring the electrokinetic potential (zeta-potential, mV) of bacterial cells was carried out on the basis of amplitude-frequency method, with the use of Zetameter-1M (Russia) in its normal operating mode (voltage — 10 V, frequency — 0.2 Hz), by measuring the oscillation amplitude of 50 bacterial cells in microelectrophoresis chamber (size: 22x22 mm, height 0.2 mm) and by calculating the average values of zeta-potential for strain according to Smo-luchowski's approximate formula [14]. The obtained results were subjected to statistical processing by methods ofvariation statistics and correlation analysis [6].
Results
The use of a single methodological technique — measuring two indicators of physicochemical properties of bacteria (hydrophil-ic-lipophilic balance and zeta-potential) — allowed us to estimate the value and direction of conversion of physicochemical properties ofbiofilm-forming bacteria resistant to the innate immunity factors (lysozyme, blood serum, low molecular weight platelet peptides).
On average, low values of hydrophilic-lipophilic balance and electrokinetic potential were typical for intact bacteria: coagu-lase-negative staphylococci (-1.441±0.12 AU; -33.4±0.4 mV, respectively), S. aureus (-1.314±0.05 AU; -32.2±0.6 mV), E. coli (-0.031±0.04 AU; -14.8±0.2 mV).
Incubation of bacteria with lysozyme led to the reduction of HLB of resistant S. aureus (AHLB = -0.65±0.08 AU) and the increase of hydrophobicity coagulase-negative staphylococci (AHLB = 0.86±0.08 AU), but did not influence the hydrophilic-lipophilic balance ofEscherichia coli (AHLB = -0.09±0.01 AU). The bacteria resistant to blood serum were more hydrophilic than intact cells. It was typical both for S. aureus (AHLB = -0.49±0.11 AU) and E. coli (AHLB = -0.02±0.03 AU).
However, coagulase-negative staphylococci resistant to blood serum were characterized on average by increase ofhydrophobicity (AHLB=0.40±0.11 AU), in comparison with intact cells.
Incubation of bacteria with a fraction of low molecular weight peptides of platelet lysate (hPL) led on average to the hydrophilization of bacterial surface. So, as compared with intact cells, bacteria E.coli (AHLB=-0.11±0.01 AU) and S. aureus (AHLB=-0.60±0.14 AU) were distinguished by a higher degree of hydrophilic surface. However, this regularity was broken only in case of coagulase-negative staphylococci resistant to antimicrobial platelet peptides. Conversion (shift) of hydrophilic-lipophilic balance of cells, caused by incubation with low molecular weight platelet peptides, contributed to the increase of hydrophobicity of resistant coagulase-negative bacteria (AHLB=0.50±0.12 AU). Selection of phenotype of staphylococci resistant to lysozyme and bactericidal activity of blood serum was accompanied by the increase of electrokinetic potential in S.aureus (Az-potential=6.80±0.64 mV; Az-potential=6.64±0.75 mV respectively), this was also typical for coagulase-negative staphylococci (Az-potential=0.87±0.43 mV; Az-potential= 2.95±0.36 mV). However, as for gram-negative cells of E. coli, incubation with lysozyme and blood serum led to the decrease of electrokinetic potential (Az-potential= -1.80±0.33 mV; Az-potential= -1.49±0.16 mV, respectively).
In general, resistance ofEscherichia coli to antimicrobial platelet peptides was consistent with the decrease of electrokinetic potential (Az-potential=-2.45±0.26 mV), this was also typical for coagulase-negative staphylococci (Az-potential=-1.22±0.24 mV). However, the resistant phenotype of S.aureus was distinguished by the increase of electrokinetic potential (Az-potential=5.37±0.58 mV). Correlation analysis was carried out in order to determine the significance of relations between the resistance to the innate immunity factors and the shift (conversion) of physicochemical properties of bacteria. The direction of HLB-shift (conversion) of biofilm-forming coagulase-negative staphylococci correlated with resistance to blood serum and lysozyme (r=0.62, p<0.05), resistance to blood serum and low molecular weight platelet peptides (r=0.43), resistance to lysozyme and low molecular weight platelet peptides (r=0.77, p<0.05). Correlation between the resistance of coagulase-negative staphylococci to the innate immunity factors and the shift of electrokinetic potential was significant only for the staphylococci, which were resistant to blood serum and lysozyme (r=0.57, p<0.05).
As for Staphylococcus aureus, which were resistant to the innate immunity factors, we noted a stronger correlation between conversion of hydrophobicity and resistance to blood serum and lysozyme (r=0.73, p<0.05). As compared to coagulase-negative staphylococci, a significantly higher correlation coefficient was revealed between the shift of electrokinetic potential of S. aureus cells resistant to blood serum and lysozyme (r=0.76), to blood serum and low molecular weight platelet peptides (r=0.60), to lysozyme and low molecular weight platelet peptides (r=0.70). Correlation of conversion direction of hydrophobicity in E.coli resistant to the innate immunity factors was significant only for bacteria resistant to lysozyme and low molecular weight platelet peptides (r=0.70, p<0.05).
Discussion
Thus, comparative analysis of conversion of physicochemi-cal properties of biofilm-forming bacteria, which are resistant to the bactericidal activity of blood serum, lysozyme and fraction of low molecular weight platelet peptides, showed that formation of resistant phenotype correlated with low hydrophilic of the bacterial surface and high values of electrokinetic potential. Conversion of physicochemical properties of bacteria during incubation with blood serum, lysozyme and fraction of low molecular weight platelet peptides is unidirectional and contributes to the decrease
of hydrophilic cells. The shift of physicochemical properties of bac- Exposure to conversion (shift) of physicochemical properties
teria increases in the range — E.coli<S.aureus<coagulase-negative of bacterial surface in interaction with the innate immunity factors is
staphylococci. typical more for staphylococci than for Escherichia coli.
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DOI: http://dx.doi.org/10.20534/ESR-17-1.2-10-12
Karimov Khamid Yakubovich, Ibragimov Zafar Zokirjonovich, Mukhamedov Rustam Sultanovich, Alimov Timur Raufovich, Boboev Kodirjon Tuxtabaevich, Scientific-Research Institute of Hematology and Blood Transfusion,
Tashkent, Republic of Uzbekistan Institute of Bioorganic Chemistry A. S. Sadykova, Academy of Sciences, Tashkent, Republic of Uzbekistan E-mail: zafar-biolog@mail.ru
Definition the frequency of gene polymorphism G2014 А ESR-a and its significance in the pathogenesis of osteoporosis
Abstract: The results of the genetic analysis showed that the frequency of ESR1 gene alleles studied in patients and controls was significantly different. It was revealed that in the basic group the association of polymorphism rs 2228480 ESR1 gene plays an important role in the osteoporosis (OP). The presence of an allele "A" and hetero/homozygous genotypes (G/A and A/A) of this polymorphism significantly increases and carrier allele genotype G and G/G lowers the risk of OP.
Keywords: single nucleotide polymorphisms, association, polymorphism rs2228480, ESR1 gene osteoporosis.
Introduction. Osteoporosis (OP) — has a complex pathogen- To date, a number of genes examined, each of which, alone,
esis is multifactorial disease characterized by low bone mass and having little effect in combination with other genes contributes
bone microstructural rearrangement, leading to increased bone to the overall phenotype of the patient. Among many candidate
fragility and risk of fracture [3; 8]. The causes of osteoporosis and genes involved in the regulation of bone metabolism, an important
osteoporotic fractures can be a genetic background, environmental role belongs to the estrogen receptor gene (ESR) [1; 2]. factors and their interaction [3; 6; 7; 9].
