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UDC: 618.12-002-022.7-085.281.9.015.8
FEATURES OF SENSITIVITY TO ANTIBACTERIAL DRUGS IN PATIENTS WITH NONSPECIFIC SALPINGOOPHORITIS
Tertyshnyk A. O.
Kharkiv National Medical University, Kharkiv, Ukraine
The features of microflora of the vagina in women patients with chronic nonspecific salpingoophoritis were studied, on the basis of which the main etiological factors of the disease, the features of the formation of associations of microorganisms which cause inflammation and the priorities for the adjustment of antimicrobial therapy were set. The investigations made indicate that in patients with chronic salpingoophoritis the changes of microflora of the vagina occur, accompanied by incoordination of its functioning as a single ecosystem, what is manifested by the disorders of microbiological status and widespread antibiotic resistance of the identified pathogens.
KEY WORDS: chronic salpingoophoritis, microflora, microbial associations, antibiotic resistance
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TepmmuHUKA. O.
XapKiBCBKiiii HaiiioHanBHiin Me/inHHiiii ymBepcHTeT, m. XapiciB, YicpaiHa
Вивчеш особливосп мжрофлори тхви у жшок хворих на неспециф1чний хрошчний салвпшгофорит, на шдстав1 чого виявлеш основш етюлопчш чинники розвитку захворювання, особливосп формування асощацш мжрооргашзм1в, яю викликаютв запалвний процес та визначеш основш напрямкн корнгування протимжробно! Tepanii. Проведен! дослщження свщчатв про те, що у пащенток з хрошчним салвпшгоофоритом вщбуваютвся змши м1крофлори nixBH, яю супроводжуютвся дискоординащею п функцюнування як едино! екосистеми, що проявляетвся порушеннями мжробюлопчного статусу та пошнреною антибютикорезистентнютю внявленнх
КЛЮЧОВ1 СЛОВА: хрошчний салвпшгоофорит, мжрофлора, мжробш асоц1аци,
ОСОБЕННОСТИ ЧУВСТВИТЕЛЬНОСТИ К АНТИБАКТЕРИАЛЬНЫМ ПРЕПАРАТАМ ПАЦИЕНТОК БОЛЬНЫХ НА НЕСПЕЦИФИЧЕСКИЙ САЛЬПИНГООФОРИТ
ТертышникА. А.
Харвковскнй нацноналвнвш медицинский университет, г. Харвков, Украина
Изученв1 особенности микрофлорв1 влагалища у женщин с неспецифическим хроническим салвпингофоритом, на основании чего вв1явленв1 основнвю этиологические факторв1 развития заболевания, особенности формирования ассоциаций микроорганизмов, которвю ввившают воспалителвнвш процесс и определенв1 основнвю направления коррекции противомикробной терапии. Проведеннвю исследования свидетелвствуют о том, что у пациенток с хроническим салвпингоофоритом происходят изменения микрофлорв1 влагалища, сопровождающиеся дискоординацией ее функционирования как единой экосистемы, проявляющейся нарушениями микробиологического статуса и обширной антибиотикорезистентноствю ввшвленнвк возбудителей.
КЛЮЧЕВЫЕ СЛОВА: хронический салвпингоофорит, микрофлора, микробнвю ассоциации, антибиотикорезистентность
INTRODUCTION
The actuality of preservation and _ rehabilitation of women's reproductive health
© Tertyshnyk A. O., 2015
has recently acquired special significance due to the increased number of chronic inflammatory diseases of the pelvic organs in women, lack of information about the pathogenic mechanisms of inflammatory reactions in presence or absence of the microbial factor, with some features of clinical course of chronic salpingoophoritis, what creates considerable difficulties in early diagnostics, with low efficiency of routine methods of treatment. Chronic inflammatory diseases of female pelvic organs are prevalent in the structure of gynecological pathology at the reproductive age with an incidence of 60-65%, and they are the cause of female infertility and menstrual dysfunctions [1, 2]. In modern conditions mixed infections of internal reproductive organs become increasingly important in the etiology of the diseases of the female reproductive system. Perhaps the change of the etiologic factors of infectious inflammatory diseases of the pelvic organs is caused by the adaptability of opportunistic microorganisms to the influence of environmentally unfavorable factors, chemical drugs, etc., by the creation of the conditions for the emergence of the strains resistant to drug influence, which acquired the complex of qualitatively new properties that make them high virulent and resistant to the influence of the immune system [3, 4].
Maintaining the stable qualitative and quantitative composition of the vaginal microbiocenosis is important in providing normal physiological status of the female organism. Normal vaginal bacterial flora is represented by different types of microorganisms, many of which are not yet identified. Quantitative bacteriological analysis of healthy women showed that 1 g of vaginal fluid contains 10s cells of aerobic and 109 cells of anaerobic bacteria. Leading microorganisms are Lactobacillus, Peptococcus, Bacteroides, Staphilococcus epidermidis, Corinebacterium spp., Peptostreptococcus spp., Eubacterium. This list represents the rank location of the dominant microbiota based on concentrations of more than 10s CFU per gram [5, 6].
It is reasonable to consider microbial consortium of vaginal mucous membranes as an organized biofilm which specifically changes into pathogenic condition that includes a set of permanent agents such as Pseudonocardia, Fusobacterium, Haemophylus, Klebsiella, Streptococcus, Staphylococcus epidermidis and
Clostridium perfringens and other periodically active community members [7].
Thus, the above-said suggests actuality of further deep study of the pathogenetic features of nonspecific salpingoophoritis on the background of identification of its microbiological features, all the more so since scientific sources suggest the lack of effectiveness of existing methods.
OBJECTIVE
The study aims to improve the treatment of the patients with nonspecific salpingoophoritis on the basis of determination of susceptibility of vaginal microflora to antibiotic drugs.
MATERIALS AND METHODS
The study evaluated the features of vaginal microflora in 70 patients aged 25 to 39 suffering from nonspecific chronic salpingoophoritis compared with the control group (35 healthy women).
The test groups were distributed as follows: group 1 - female patients with a history of chronic salpingoophoritis lasting up to 10 years (n = 35), group 2 - patients with chronic salpingoophoritis lasting more than 10 years (n = 35); group 3 - almost healthy women (comparison group).
For the assessment of the microorganism content in genital secretions of women the tested material was taken from the posterior vaginal vault and subjected to bacteriological examination. The microflora was evaluated by the method of H. Haenel (1979) in the modification of S. K. Kanareykina (1981), under which the following was taken into account: 1) the frequency of occurrence of the microorganisms in this biotope; 2) general dissemination; 3) the quantity and type composition of: a) lactobacilli; b) streptococci; c) staphylococci; d) enterobacteria; e) fungi of the genus Candida; 4) microbial associations [6]. Removal of isolates from vaginal secretions and cervical scrape was made by the conventional methods in microbiology. Enzymatic identification was made with the help of identification sets MICRO-LA-TEST®, designed for providing standard identification using micro methods. They allow identification of most clinically important microorganisms in short terms.
The sensitivity of the isolates to antimicrobial agents with different mechanisms of action on a microbial cell was studied by
means of microtest system «TNK test» with semiquantitative registration of results.
The data were processed after building the databases in Microsoft Excel, Statistica 7.0. For statistical evaluation of the results parametric criteria were used (mean - M, standard deviation sd) and non-parametric criteria (absolute (n, number) and relative (percentage (p, %) and the average error of rate (sP), criterion %2) units). Statistical reliability of differences between groups was evaluated by nonparametric U-Mann-Whitney test. The results were considered adequate at the significance level p < 0,05 and p < 0,01.
RESULTS AND DISCUSSION
As a result of the study of the female patients of groups 1 and 2 violations of the vaginal microbiocenosis were found. Analyzing the obtained data, we can prove that the most frequent microorganisms removed from the vaginal discharge were: Peptostreptococcus spp - 78 % and 75 %, Enterococcus - 69 % and 57 %, S.aureus - 62,9 % and 60,1 %, E.coli -64,2 % and 69,3 %, Fusobacterium spp -61,0 % and 58.0 %; ,S'. pyogenes - 58,0 % and 60 %, Candida spp - 47,0 % and 44 %.
During bacteriological study from biomaterial the isolates were typically removed in associations (Fig.).
S. pyoge nes+Pe ptostre ptococc us+C.albicans
S. aureus+ Enterococcus+
E.coli+ C.albicans E.coli+ P.aeruginosa+ S.
aureus+ Proteus spp.
P.aeruginosa+
5. pyoge nes+Pe ptostre ptococc
us+C.albicans
C.albicans+ S.pyogenes+
E.coli+Bacteroides
Citrobacter spp.+ C.albicans
S.pyogenes+
Peptostreptc^tefe!jfllasPP
Pe ptostre ptococcus+
Enterococcus
spf
S.pyogenes+ Proteus Pe ptostre ptococcus
E.coli+ S. aureus+ Bacte
eptostre ptococcus+
S.pyogenes+ E.coli ptostreptococcus+ S.
aureus+ S.pyogenes
S. aureus+ E.coli+ Pe ptostre ptococcus
Fig. Associations of isolates separated in salpingoophoritis
Among the coccal flora there were staphylococci, which were found in the patients of two test groups. Dissemination with staphylococci was averaged 1,7-105 ± 1,2-104 and 4,1-104± 1,5 103 CFU per unit substrate respectively, the most frequent was S.aureus with the density of microbial colonization
6,3104± 1,2-103 and 7,5-104± 1,3-103 CFU per unit substrate respectively. Microbiocenosis of anaerobic flora in patients was diverse. Thus, in the vaginal microflora of the test groups Veillonella spp and Prevotella spp. were found with the dissemination equal to 1,8-104± 1,2-104 and 2,9-104± 1,6-103, and
1,5-104± 1,6-104and 3,7-103± 1,4-102 CFU per unit substrate. Bacteroides spp was detected with dissemination of 4,4-102± 1,8-101 and 1,3-103± 1.6-102 CFU per unit substrate. At the same time in the microbiocenosis of vagina Propionibacterium spp. was registered. Its colonization density was equal to 1,9-10s± 1,6-104 and 5,7-104± 1,2-103 CFU per unit substrate in accordance.
The results of the comparative evaluation of the sensitivity to antimicrobial drugs of
therapeutic purpose of the isolates which were more frequently disseminated in patients with salpingoophoritis (staphylococci, streptococci, enterobacteria and obligate anaerobes) showed that the incidence of identification of the isolates of Staphylococcus resistant to antimicrobial drugs was much different and depended on the duration of an inflammatory process (tab. 1).
Table 1
Antibiotic resistance of Staphylococcus isolates
Antibiotics Rate of resistant strains (M ± м)
Group 1 Group 2 Group 3
Cefepime 5,6 ± 5,4 4,4 ± 16,6*# 2,0 ±3,3
Ceftazidime 11,1 ±7,4 7,0 ±6,2* * 3,0 ±3,3
Ciprofloxacin 12,0 ±3,6 11,1 ±2,5** 7,0 ±3,3
Rifampicin 76,0 ± 3,6 44,4 ±16,6* * 31,0 ±3,3
Erythromycin 88,9 ±7,5 91,2 ±6,2* 59,0 ± 1,2
Vancomycin 15,0 ±3,6 13,0 ±6,2** 9,0 ±3,3
Lincomycin 77,7 ± 2,8 94,4 ± 6,6* 50,0 ± 5,2
Gentamicin 91,1 ±5,5 91,3 ±6,2* 69,0 ±3,3*
Amikacin 10,0 ±3,6 11,1 ±2,5* 2,0 ±3,3
Kanamycin 93,3 ±4,1 93,0 ± 6,2* 57,0 ± 4,9
Ampicillin 61,1 ±4,5 92,8 ±6,2* * 50,0 ± 5,2
Oxacillin 95,6 ± 5,4 96,3 ± 2,2* 70,0 ± 3,3
Penicillin 72,2 ±10,6 97,2 ±1,2* • 50,0 ± 1,2
Amoxicillin 90,0 ± 3,6 93,1 ±2,2* 40,0 ± 3,3
Chlorhexidine 6,0 ±1,6 4,9 ± 0,6* Ш 1,0 ±0,3
Note: *p<0,001; **p<0,01; #p<0,05 compared with the control group (group 3); * p<0,001; II p<0,05 compared with the patient group 1.
Staphylococcus isolates were characterized by variable sensitivity to the studied drugs. The most resistant to antimicrobial drugs Staphylococcus strains were taken from the patients of groups 1 and 2. Regarding penicillin the percentage of resistant strains in the patients of group 2 was 97.2 %; the percentage of majority of cephalosporins - from 2.0 % to
11.1 %. Generally, most Staphylococcus isolates were poly-resistant.
As shown in Table 2, among Enterobacteria extracted from the patients of test groups, the rate of resistant strains was high in all groups of study. Before cephalosporins resistant strains percentage was higher for Enterobacterium isolates from the patients of group 1 - 9.3 to 16.7 %.
Table 2
Antibiotic resistance of Enterobacterium isolates
Antibiotics Rate of resistant strains (x ± Sx)
Group 1 Group 2 Group 3
Cefepime 11,3 ±0,8 16,7 ± 1,0** 2,0 ±0,3
Ceftazidime 9,3 ± 0,8 6,7 ± 1,0*® 3,0 ±0,3
Ciprofloxacin 81,3 ±8,8 86,7 ± 7,0* 7,0 ±0,3
Rifampicin 56,3 ± 2,4 96,2 ± 1,8** 31,0 ±3,3
Gentamicin 93,8 ±6,1 97,8 ± 6,2* 69,0 ±3,3
Amikacin 93,8 ±6,1 93,0 ± 1,8* 2,0 ±0,3
Kanamycin 81,3 ±8,8 88,9 ±4,7* 57,0 ± 4,9
Ampicillin 81,3 ±8,8 96,0 ±1,8*® 50,0 ± 5,2
Amoxicillin 97,0 ± 2,0 98,0 ± 1,8# 83,3 ±6,8
Carbenicillin 75,0 ±6,8 88,9 ± 4,7 73,3 ±6,8
Levomycetin 98,0 ±1,0 98,2 ± 1,8* 24,0 ± 2,5
Chlorhexidine 5,0 ±3,6 4,4 ± 0,6 1,0 ±0,3
Note: *p < 0,001; #p < 0,05 compared with the control group; * P < 0.001; 11 p < 0.05 compared with group 1
The data in Table 3 indicate that in patient strains of streptococci ranged from 11.0 % to groups 1 and 2 of the number of resistant 98.6%.
Table 3
Antibiotic resistance of Streptococcal isolates
Antibiotics Rate of resistant strains (x ± Sx)
Group 1 Group 2 Group 3
Clindamycin 69,0 ± 2,5 28,6 ±2,1** 1,0 ±0,15
Erythromycin 73,3 ±7,6 88,6 ±2,1** 10,0 ±0,9
Ceftriaxone 15,0 ± 1,1 17,7 ± 1,4* 9,0 ± 0,2
Cefotaxime 16,7 ± 1,8 15,7 ± 1,4* 8,0 ±0,9
Ampicillin 93,3 ± 2,6 98,6 ± 1,1* 50,0 ± 5,0
Azithromycin 90,0 ±2,1 88,6 ±2,1* 5,0, ± 2,8
Vancomycin 11,0 ± 1,5 11,0 ± 1,1* 1,0 ±0,1
Levomycetin 86,7 ± 6,8 92,85 ± 3,2* 24,0 ± 2,5
Ofloxacin 28,9 ± 2,5 28,6 ±2,1* 1,0 ±0,15
Sisomicin 12,0 ± 1,5 14,3 ±1,4 15,0 ± 1,6
Chlorhexidine 13,0 ±3,6 14,4 ±4,6*® 1,0 ±0,3
Note: * p < 0.001; ** p < 0.01; # P < 0.05 compared with the control group; * P < 0,001; • p < 0,01; a p < 0.05 compared with group 1.
Evaluation of the resistance of the isolates G -» and «TPK G + » (tab. 4) showed that all to antimicrobial agents using microplate «TPK strains were variable to antimicrobial drugs and
most strains were resistant to ampicillin and doxycycline and moderately resistant to gentamicin. The analysis of the data obtained allowed to find out that all isolates had multiple antibiotic resistance. The studies
showed that the isolates were resistant to ampicillin, gentamicin and doxycycline - the growth of culture was observed in the microplate cells with higher and lower concentrations of antimicrobial preparations.
Table 4
The sensitivity of isolates to antimicrobial drugs inoculated in microplate cells «TPK G -» and «TPK G + »
Antimicrobial drugs inoculated in microplate cells «TPK G -» and «TPK G + » Staphylococci Streptococci Enterobacteria
R % I % S % R % I % S % R % I % S %
Cefotaxime 4 4 92 4 7 89 80 9 11
Ciprofloxacin 11 1 88 30 4 66 86 2 12
Gentamicin 94 6 0 97 3 0 97 3 0
Ampicillin 69 31 0 93 6 1 98 2 0
Doxycycline 96 4 0 94 6 0 88 12 0
Thus, in determining the susceptibility of isolates to antibiotics it has been discovered that most of them were multiresistant (89.2 %). Variable sensitivity to antimicrobial agents related to glycopeptides, fucidins, rifampicin and lincozamides was observed.
Thus, the development of the diseases of microbial etiology depends on the persistent properties of microorganisms aimed for the inactivation of the factors of natural resistance of the organism. The type composition and biological properties of the vaginal microflora in women with nonspecific salpingoophoritis were studied and it was shown that inflammatory diseases of internal reproductive organs occur on the background of dysbiotic disorders characterized by the release of microorganisms with high persistent properties. The latter obviously play an important role in the pathogenesis of inflammatory diseases and dysbiotic conditions of internal reproductive organs.
Based on the above it was confirmed that the occurrence and intensity of inflammatory process depend on the individual fluctuations in qualitative and quantitative composition of microflora of the vagina.
The main practical problem of the diagnosis verification primarily concerns timely determination of the cause of inflammatory process and is associated with the demand of accurate typing of the etiological factor of salpingoophoritis. During the detection of
microbial associations the difficulties which arise during the treatment of patients with salpingoophoritis have become clear. The first reason of any failure is persistent diagnostics of the etiological factors while ignoring the pathogenic potential of normal microflora. The second reason is common difficulties in transporting the drugs to the focus of inflammation. The third cause little known to clinicians is group resistance of antibiotics and other effects (quorum sensing) of locus microbiota organized in biofilm.
From the overview of the information on the distribution of microbial associations during the inflammation of the pelvic organs, their multiple antibiotic resistance, antibiotic drugs should be administered immediately after clarifying the nosological diagnosis and before getting the results of bacteriological research. After receiving the results of the bacteriological study the mode of antimicrobial therapy should be adjusted for the selected microflora and its antibiotic sensitivity. Therefore, adequate microbiological diagnosis of ascending infection which causes development of nonspecific salpingoophoritis should be given enough attention as well as to the choice of a mode of therapy.
PROSPECTS FOR FUTURE STUDIES
The current stage of the development of medicine is characterized by insufficient effectiveness of therapy of ascending infection
that leads to salpingoophoritis and then to its chronic course. To a certain extent this is explained by to the presence of mechanisms of protection of pathogens from damaging factors. One of such mechanisms is the ability to form biofilm. Therefore, the study of the ability to
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