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K.M11MIIA ME.UMUIMA
CMMCAL MEDICINE
DOI 10.26724/2079-8334-2019-2-68-7-11 UDC 616.23/24-057:575.113.1
T A. Anili iishilnnko '. D.O. Stroy S.V . Goncharov V .E. Dosenko 2. K.E. Ishhejkin ' 1 State Institution "Kundiiev Institute of Occupational Health of the National Academy of Medical Sciences of I kraine". Kyiv. 2 Bogomolet/ Institute of Physiology of the National Academy °l Sciences ol I k|aine. Iwn. I k|ainian medical stoma|o|ogila| academy. I °hava
GENETIC PREDISPOSITION TO BRONCHOPULMONARY PATHOLOGY
E-mail: imp-cys@ukr.net
We studied 215 people who work in harmful industries, 90 of which had a history of bronchopulmonary pathology of occupational etiology and 125 of them without such pathology. The following polymorphisms of DNA repair genes were identified in real-time using polymerase chain reaction: XPD (rs13181, rs799793), ERCC1 (rs11615), XRCC3 (rs861539), XRCC1 (rs25487), ATM (rs664677), XRCC7 (rs7003908) and MLH1 (rs1799977). We studied the frequency distribution of the genotypes of DNA repair genes with the subsequent integral statistical analysis of the data obtained. Analysis of the results made it possible to build a mathematic model that included two single nucleotide polymorphisms: XRCC1 (rs25487) and ATM (rs664677), which in this study represented the two main independent effects with the greatest predictive power 80.35 % for the results of binary logistic regression and of the method of multivariate dimension reduction.
Keywords: SNP:XPDrERCCLXRCC3rXRCCLATM, XRCC7.MLHL bronchopulmonary pathology.
The work is a fragment of the research project "Genetic markers that measure efficacy ofDNA repair under the influence of occupational factors" (state registration №0119U101613).
In Ukraine 6-8 thousand professional diseases are registered annually, of these 70% cause bronchopulmonary pathology (BPP) [2]. The group of endogenous factors that determine its development BPP individually determined; the activity of the enzymes that are responsible for the metabolism and polymorphism of genes is genetically determined, SNP, they regulate immune response, apoptosis, cell regeneration, etc. [1]. In living organisms, there are various systems that protect from exogenous damaging agents, such as DNA repair [3, 6, 8, 9, 14].
Up to 70% of DNA damage is eliminated by BER (base-excision repair) proteins [8]. The existence of polymorphisms of BER genes increase the frequency of chromosomal aberrations, discontinuities, DNA adducts, and micronuclei [1]. XRCC1 (X-ray-repair cross-complementing group 1) encodes a protein that regulates the regeneration of DNA molecules from damage by ionizing radiation and by alkylating agents [6, 14]. XRCC3 (X-ray-repair cross-complementing group 3) is involved in recombinant DNA repair and double-stranded DNA breaks [11]. Proteins of NER (nucleotide excision repair) genes are responsible for removal of damaged nucleotides through the recognition and correction of basal cross-links [1]. The XPD protein (xeroderma pigmentosum group D) functions at the beginning of the synthesis of all proteins in the complex protein TFIIH [3, 8]. The main function of ERCC1 (Excision repair cross complementing 1) is nucleotide recovery, allele ERCC1 118T is associated with a decrease in mRNA and a decrease in NER activity [1]. DSBR (double-strand break repair) error variants lead to various mutations and chromosome rearrangements that induce genome instability and carcinogenesis [8]. XRCC7 (X-ray-repair cross-complementing group 7) encodes a protein that is a large catalytic subunit of the initiating DNA-PKc complex [6, 7, 15]. The ataxia-telangiectase mutation (ATM) gene encodes for DNA-dependent proteinurase, which participates in the mitogenic signal of meiotic recombination and in the regulation of the cell cycle [12]. MMR (mismatch repair) stores genetic information under conditions that increase the frequency of mutations. MLH1 (mutL (E.coli) homolog 1) encodes a protein that regulates the replacement of improperly coupled DNA bases and is inactivated by methylation [10, 12].
However it is now clear that the end result is the consequence of the work of many genes and modifying factors, therefore many researchers have chosen to study the influence of highly penetrating polymorphisms on the chemical carcinogenesis of a person who smokes tobacco. In this study, we researched the propensity of molecular genetic markers to predict the development of BPP in persons operating under conditions of industrial aerosols. It is known that the nature, clinical course and complications of BPP are determined by the chemical composition of the industrial aerosols, the quantity of aggressive substances contained therein and the individual characteristics of the organism.
The purpose of the work was to determine the predictors of the risk of developing BPP in people who work in harmful industries.
© T.A. Andrushchenko, D.O. Stroy, 2019
ISSN2079-8334. Ceim Meduu,UHU ma dio^ii. 2019. № 2 (68)
Materials and methods. All procedures performed in the study involving the people meet the ethical standards of the Bioethics Commission of the State Institution "Institute of Occupational Health of the National Academy of Medical Sciences of Ukraine ", February 29, 2016). All respondents agreed to voluntarily participate in the study and sign informed consent.
Characteristics of people included in the study. Employees of harmful industries (coal miners and workers of asbestos cement plants (ACP)), who worked with industrial aerosols (n = 215) were included. The experimental group (n = 90) included workers from ACP and coal miners with BPP (chronic bronchitis, chronic obstructive pulmonary disease, pneumoconiosis) ages 22 to 84 years, with an average age of 50.5 ± 7.3 yrs., average time of exposure to harmful substances 21.0 ± 6.1 yrs., the average age of starting employment in above started industry is 28.7 ± 6.8 yrs.. The control group (n = 125) included workers ages 20 to 80 yrs. old;, their average age was 45.0 ± 7.2 yrs., the average time of exposure to harmful substances 16.9 ± 5.4 yrs., the average age of starting employment in above started industry is 26.4 ± 6.7 yrs.
Blood samples for genotyping were collected in sterile conditions in 2.7 ml of monovets containing potassium salt of EDTA ("Sarstedt", Germany), followed by the freezing of specimens and their storage at -20 ° C. DNA for genotyping was isolated using NeoPrep100DNA and NEOGENE (Ukraine) kits in accordance with the manufacturer's instructions. Real-time polymerase chain reaction was performed on the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, USA) using TaqMan Assays for polymorphisms: XPD (rs13181 and rs799793), ERCC1 (rs11615), XRCC3 (rs861539), XRCC1 (rs25487), ATM (rs664677), XRCC7 (rs7003908) and MLH1 (rs1799977).
Statistical analysis. Clinical data was analyzed for the normality of the distribution using the Shapiro-Vilka test, as well as by the Levine Leuven test, the assumption of the equality of dispersions was checked, after which Student's statistical criterion (statistically significant results calculated P < 0.05). All calculations were performed on the basis of SPSS ver.17.0, ver.23. The SNP Analyzer program was used to verify the Hardy-Weinberg equilibrium. The main independent and consistent effects of all analyzed polymorphisms were determined by the following statistical methods: logistic regression, Multifactorial Dimensionality Reduction (MDR) to evaluate both the independent and consistent effects of the eight analyzed DNA polymorphisms analyzed in order to construct a mathematical model.
Results of the study and their discussion. In the first stage of our study, associations of polymorphisms of DNA repair genes with the risk of developing BPP in patients and healthy individuals who worked in harmful conditions were analyzed. It was found that the distribution of 4 of the 8 polymorphisms of the DNA repair genes studied significantly differed between the respondents of the experimental and control groups: XPD (rs13181), XRCC1 (rs25487), ATM (rs664677) and MLH1 (rs1799977) (table 1).
Table 1
Distribution of genotypes of polymorphisms of DNA repair genes (hi-square, odds ratios, confidence intervals
polymorphisms genotypes Value of P, x2 OR, 95% CI
XPD (rs13181) AA P = 0.050 1.03 (0.56 - 1.88)
Aa 0.61 (0.34 - 1.09)
aa 2.20 (1.02 - 4.77)
XPD (rs799793) AA P = 0.700 0.81 (0.45 - 1.46)
Aa 1.10 (0.62 - 1.97)
aa 1.28 (0.48 - 3.44)
ERCC1 (rs11615) AA P = 0.800 0.95 (0.53 - 1.71)
Aa 1.16 (0.64 - 2.09)
aa 0.83 (0.39 - 1.79)
XRCC3 (rs861539) AA P = 0.900 0.98 (0.54 - 1.76)
Aa 0.98 (0.55 - 1.75)
aa 1.10 (0.44 - 2.75)
XRCC1 (rs25487) AA P = 0.010 1.13 (0.63 - 2.04)
Aa 0.57 (0.32 - 1.02)
aa 3.37 (1.22 - 9.63)
ATM (rs664677) AA P = 0.030 0.83 (0.45 - 1.54)
Aa 0.67 (0.38 - 1.21)
aa 2.48 (1.16 - 5.31)
XRCC7 (rs7003908) AA P = 0.700 1.08 (0.60 - 1.93)
Aa 1.06 (0.59 - 1.91)
aa 0.74 (0.30 - 1.81)
MLH1 (rs1799977) AA P = 0.008 0.43 (0.24 - 0.79)
Aa 2.32 (1.29 - 4.21)
aa 0.97 (0.32 - 2.91)
ISSN2079-8334. Ceim Meduuuuu ma ôio^iï. 2019. № 2 (68)
The next step in the study was to use logistic regression and MDR techniques, to build a mathematical model with the greatest potential for prediction. It included the following predictors: yrs. working in harmful industry; yrs. wich started employment in industry; findings from the manual review of the radiographs of the chest organs; family history of BPP (clinical genealogy analysis of pedigrees); smoking status; polymorphism XRCC1 (rs25487); polymorphism ATM (rs664677) with a prediction potential of 83.3%. Regression coefficients, 95% confidence intervals, values of P are shown in table 2.
Table 2
Significance of predictors based on the results of logistic regression
Variables B d.f. P Exp(ß)
Time of employment in harmful industry, yrs. 0. 038 1 0.010 1.456
Age at start of employment, yrs. 0.046 1 0.038 1.564
XRCC1 1 (Aa) 0.750 1 0.867 0.928
XRCC1 2 (aa) 2.186 1 0.005 8.897
ATM 1(Aa) 0.482 1 0.308 1.620
ATM 2 (aa) 1.205 1 0.023 3.335
Basal pneumosclerosis 3.926 1 0.001 50.723
Basal and roar pneumosclerosis 24.678 1 0.999 -
Diffused pneumosclerosis 3.559 1 0.001 35.119
Family history BPP 0.827 1 0.001 2.300
Smokes - 0.457 1 0.001 1.894
Does not smoke - 1.192 1 0.158 0.304
Constant - 5.850 1 0.000 0.003
Note: the regression coefficients are: p - coefficient of binary logistic regression; d.f. Exp (p) is the odds ratio (OR).
Degrees of freedom; P - statistical significance;
The use of the MDR method confirmed that there is a high-power synergistic interaction between the polymorphisms XRCC1 (rs25487), ATM (rs664677), XPD (rs13181) and MLH1 (rs1799977) and they are in close correlation with the other genetic polymorphisms studied (fig. 1).
The strongest effect was exhibited by polymorphism MLH1 (rs1799977) -3.25 %; the second strongest by XRCC1 (rs25487) - 2. 84 %; the third by ATM (rs664677) - 2.23 %; XPD (rs13181) - 1.91 % entropy (Fig. 1).
In analyzing the frequency of genotypes of DNA repair genes, it was found that the heterozygote XRCC1*G/A (rs25487) and the dominant homozygote MLH1-A/A (rs1799977) have a protective effect, while the genotypes XPD'C/C (rs13181), XRCC1AA (rs25487), ATM'T/T (rs664677) and MLH1-A/G (rs1799977) are associated with the risk of BPP. The obtained data require further validation, study and comparison with results obtained by other studies. These polymorphisms were previously considered by researchers as markers of carcinogenesis of various types and localizations, including lung cancer, and most studies were simulated on smokers. Since tobacco smok has a multicomponent chemical composition that causes all possible damage of DNA [13]. The value of even unpredictable variants of SNP and their protein products involved in DNA repair has been established. In particular, smokers with a high level of induction of CYP1A1 and CYP2D6, which was detected in lymphocyte cultures when treated with polycyclic aromatic hydrocarbons, had a significantly higher incidence of lung cancer [5, 13]. And if smoking was combined with pneumoconiosis from asbestos dust, the risk of developing lung cancer increased 18-fold [4].
Inclusion in the analysis of non-genetic predictors has allowed to improve the predictive value of the logistic regression mathematical model and to develop an algorithm for determining the propensity to
Fig. 1. Dendrogram of intergenic interactions. Red is the synergistic interaction of high power, orange - the synergetic interaction of medium power, brown - independent main effects, green - the antagonistic interaction of medium power, blue color - antagonistic interaction of significant strength.
develop BPP among workers of harmful industries, which in turn will allow to improve the performance of preliminary medical examinations when hiring in such harmful industries and the quality of periodic medical examinations of those, who are already working.
Conclusion
For the first time results indicating the significance of polymorphisms of DNA repair genes in the formation of predisposition or resistance to the development of bronchopulmonary pathology among workers of hazardous and harmful industries of Ukraine were obtained. Established genotypes associated with the risk of developing bronchopulmonary disease: XPD'C/C (rs13181) (OR = 2.20; 95% CI: 1.02 -4.77; P = 0.020; x2 = 4.85);XRCC1AA (rs25487) (OR = 3.37; 95% CI: 1.22 - 9.63; P = 0.008; x2 = 6.94); ATM'T/T(rs664677) (OR = 2.48; 95% CI: 1.16 - 5.31; P = 0.010; X2 = 6.61);MLHhA/G (rs1799977) (OR = 2.32; 95% CI: 1.29 - 4.21; P = 0.002; x2 = 9.01). Also identified were genotypes that promote resistance to the development of respiratory diseases: XRCC1G/A (rs25487) (OR = 0.57; 95% CI: 0.32 - 1.02; P = 0.040; x2 = 4.14); MLH1AA (rs1799977) (OR = 0.43; 95% CI: 0.24 - 0.79; P = 0.003; x2 = 8.73).
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Реферат
ГЕНЕТИЧНА СХИЛЬШСТЬ ДО БРОНХОЛЕГЕНЕВО1 ПАТОЛОГИ Андрущенко Т.О., Строй Д. А., Гончаров С.В., Досенко B.C., IiueiiKiN К.С.
У 90 пращвниюв шюдливих галузей промисловостЦ в анамнезу у яких бронхолегенево'' патологи професшно'' етюлогп i 125 пращвниюв аналопчних професш за допомогою полiмеразноï ланцюгово'' реакцп в реальному чаа були визначеш наступш полiморфiзми гешв репарацп ДНК: XPD (rs13181, rs799793), ERCC1 (rsl 1615), XRCC3 (rs861539 ), XRCC1 (rs25487), АТМ (rs664677), XRCC7 (rs7003908) i MLH1 (rs1799977). Вивчали розподш частот генотитв гешв репарацп ДНК з подальшим штегральним статистичним аналiзом отриманих даних. Аналiз результата дав можливють побудувати математичну модель, яка включала в себе два однонуклеотидних полiморфiзми: XRCC1 (rs25487) i
ГЕНЕТИЧЕСКАЯ СКЛОННОСТЬ К БРОНХОЛЁГОЧНОЙ ПАТОЛОГИИ Андрущенко Т.А., Строй Д.А., Гончаров С.В., Досенко В.Е., Ищейкин К.Е.
У 90 работников вредных отраслей промышленности в анамнезу у которых бронхолёгочная _налiз_м_ профессиональной этиологии и 125 работников аналогичных профессий при помощи полимеразной цепной реакции в реальном времени были определены следующие полиморфизмы генов репарации ДНК: XPD (rs13181, rs799793), ERCC1 (rs11615), XRCC3 (rs861539), XRCC1 (rs25487), АТМ (rs664677), XRCC7 (rs7003908) и MLH1 (rs1799977). Изучали распределение частот генотипов генов репарации ДНК с _налiз_м_им интегральным статистическим _найз_м полученных _налiз. _налiз результатов дал возможность построить математическую модель, которая включала в себя два однонуклеотидных полиморфизма:
АТМ (rs664677), що е в даному дослщженш двома головними незалежними ефектами з найбшьшою предиктивно! силою 80,35% за результатами бшарно! лопстично! регресп i методу багатофакторного зменшення розмiрностi.
Ключовi слова: однонуклеотидний полiморфiзм; XPD, ERCC1, XRCC3, XRCC1, АТМ, XRCC7, MLH1, бронхолегенева патологiя.
Стаття надiйшла 19.06.18 р.
XRCC1 (rs25487) и АТМ (rs664677), являющиеся в данном исследовании двумя главными независимыми эффектами с наибольшей предиктивной силой 80,35 % по результатам бинарной логистической регрессии и метода многофакторного уменьшения размерности.
Ключевые слова: однонуклеотидный полиморфизм; XPD, ERCC1, XRCC3, XRCC1, АТМ , XRCC7, MLH1, бронхолёгочная патология.
Рецензент Костенко В.О.
DOI 10.26724/2079-8334-2019-2-68-11-15 УДК: 616.37-002-036.11-022-085.33
ШшшшшМшШ
В. 11. Андрюшенко Д.В. Ан дрюшенко. В. А. Mai.и.ованим
ХАРАКТЕРИСТИКИ СУЧАСНО1 МАНКРЕАТИЧНО1 1НФЕКЦН ТА IX ЗНАЧЕННЯ В ОБГРУНТУВАНН1 АНТИБЮТИКОТЕРАМН МРИ ГОСТРОМУ ПАНКРЕАТИТ
E-mail: avp.victor@gmail.com
З метою з'ясування характеристик сучасно! панкреатично! тфекцп (П1) на першому етат роботи проведено 147 бактерiологiчних дослщжень матерiалу - вмiсту перипанкреатичних рщинних скупчень та фрагментiв некротизованих тканин тдшлунково! залози i заочеревинно! ^тковини у 44 хворих на гострий панкреатит (ГП). Констатовано, що П1 була представлена мкрофлорою з домшуванням аеробних бактерiй у виглядi монокультури (37%) та аеробно-анаеробних асощацш (39%) з грам-негативною ознакою (56%), рiзною тропнiстю бактерiй до патоморфологiчних субстраив та незмiннiстю iснуючих асоцiацiй у 57% оЫб. Встановлено чутливiсть П1 до рiзних груп антибiотикiв. На другому етат дослщження у 460 хворих на ГП ощнено ефективтсть та визначено доцiльнiсть реалiзацil рiзних режимiв антибютикотерапп, зокрема превентивно!, яка сприяла збереженню асептичностi запального процесу у 68% спостережень та етютропно! - при шдукованш полiрезистентностi мжрофлори.
Ключов! слова: гострий панкреатит, панкреатична ¡нфекщя, режими антибютикотерапп.
Робота е фрагментом НДР «Опрацювання дiагностичних та л^валъних nidxodie при гострому naHKpeamumi та гншно-некротичних ураженнях м 'яких тканин з використанням клШчних, лабораторно-бiохiмiчнuх, морфологiчнuх, променевих та шструменталъних методик обстеження» (державний реестрацшний № 0115 U00343).
Гострий панкреатит (ГП) являе собою одну з найбшьш актуальних проблем невщкладно! абдомшально! х1рургй [2, 6, 10, 12 ]. Сучасний тдхщ до виршення И полягае у опрацюванш мультидисциплшарного принципу лшувально! тактики, який вщдзеркалюе загальну тенденщю медицини стосовно штеграцй та взаемовпливу р1зних спещальностей при вивченш складних бюлопчних об'екпв [2, 3, 8, 11 ].
Одним 1з прюритетних компоненпв зазначеного тдходу при ГП е з'ясування характеристик сучасно! панкреатично! шфекцй (П1) з оцшкою клшчного значення !! для виршення прюритетних питань лшувально! тактики i, зокрема, доцшьносп та оптимальних режишв призначення антибактершних медикаментозних засоб1в - антибютиюв в процес л1кування хворих [5,7,10,13]. Тому необхщшсть виршення даного завдання набувае зростаючого значення.
Метою роботи було з'ясування особливостей сучасно! П1 з поглибленим анатзом !! основних характеристик та оцшкою значення отриманих результапв для обгрунтування режиму антибютикотерапп' при ГП.
Матер1ал та методи дослщження. Дизайн дослщження полягав у реатзацп двох послщовних етатв. На першому проводилось з'ясування характеристик сучасно! П1, на другому -здшснювався анал1з ефективносп застосування антибютиюв в процес1 лшування хворих. Мшробюлопчну складову роботи виконано в процес л1кування 44 пащенпв на ГП у вщ1 в1д 34 до 68 роюв (середнш в1к - 50±5,2 ). Чоловшв було 29 (67%), жшок -15 (37%). Для вивчення та оцшки бактершного чинника (БЧ), який проявлявся в клшчному перебпу захворювання з локал1защею в перипанкреатичних рщинних скупченнях (РС) i тканинах тдшлунково! залози та заочеревинно! клггковини, здшснено 147 бактерюлопчних дослщжень (БД) матер1алу з проведенням аеробного i анаеробного способ1в його культивування при забор! шд час проведення мшмально швазшних операцшних втручань i традицшних лапаротомних операцш. Дослщження виконане на кафедр! мшробюлогй та в1русологй Льв1вського нацюнального медичного ушверситету 1меш Данила Галицького.
© В.П. Андрющенко. Д.В. Андрющенко, 2019
