DOI 10.1007/s10517-015-2857-1
766
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 PHARMAСOLOGY AND TOXICOLOGY
Effects of Antibiotic Treatment on the Lactobacillus Composition of Vaginal Microbiota A. R. Melkumyan, T. V. Priputnevich, A. S. Ankirskaya, V. V. Murav’eva, and L. A. Lubasovskaya Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 12, pp. 730-733, December, 2014 Original article submitted April 21, 2014 We analyzed sensitivity of 123 vaginal lactobacillus strains to antibacterial substances. All lactobacillus strains were sensitive to ampicillin, cefazolin, cefotaxime, and vancomycin, and insensitive to metronidazole, trimethoprim/sulfamethoxazole, and levofloxacin. Lactobacillus strains demonstrated different sensitivity to gentamycin, clindamycin, erythromycin, ciprofloxacin, and tetracycline. The phenomenon of preferential selective influence of antibacterial drugs on the composition of lactobacilli of the vaginal microbiota, in which some lactobacilli survive as part of the vaginal microbiota and have a selective advantage over other types of lactobacilli, should be taken into account during treatment of vaginal infections and dysbiosis. Key Words: vaginal microbiota; lactobacilli; L. crispatus; L. iners; antibiotic sensitivity Effects of antibacterial substances (ABS) on the resident component of vaginal microbiota during treatment of dysbiosis and inflammatory processes in the urogenital system have two points. Minimization of the effects of ABS on normal microflora can induce resistance of “useful” microflora to the used ABS. However, during long-term and frequent antibiotic therapy the normal microflora can obtain certain mechanisms of resistance to antibiotics and transfer resistance genes to pathogenic microorganisms and transitory microflora. The problem of administration of probiotics in parallel with antibiotic therapy is actively discussed in literature. Under these conditions, antibiotics affect not only pathogenic microorganisms, but also useful probiotic strains [1,3,4]. Standardization of the methods for estimation of phenotypic resistance of bacteria from normal microflora is a necessary condition for recognizing genotypes resistant to ABS. Current traditional standardized methods for estimation of the sensitivity of clinically significant bacteria can hardly be extrapolated to V. I. Kulakov Research Center for Obstetrics, Gynecology, and Perinatology, Ministry of Health of the Russian Federation, Moscow, Russia. Address for correspondence: alinamelkumyan@yandex. ru. A. R. Melkumyan
non-pathogenic bacteria, e.g., lactobacilli. Difficulties in evaluation of antibiotic sensitivity of Lactobacillus strains isolated from the blood of patients with endocarditis showed the necessity of development of standards for estimation of sensitivity of lactobacilli with consideration for their growth requirements and distribution in antibacterial medium. Several investigations for standardization of estimation of lactobacillus sensitivity were performed previously [1,4,6,9]. Various methodological approaches, nutrient media, regimens, and culturing terms and conditions were used in these experiments. Within the framework of EC ACEART project, standard operational procedures for estimation of the sensitivity of lactobacilli to antimicrobial substances using disk diffusion test (DDT), E-tests, and serial dilution method (SDM) in broth were developed. Experiments [8] showed that each method has advantages and disadvantages. “Agar” methods, e.g., E-test and DDT (especially E-test), have some advantages over SDM. Antibacterial resistance safety problems are certainly taken into account during development and marketing of probiotics. Resolution of the Scientific Committee on Animal Nutrition (SCAN; 2001) of Europe stated criteria for estimation of safety of microorganisms insensitive to antibiotics for the use as probiot-
0007-4888/15/15860766 © 2015 Springer Science+Business Media New York
A. R. Melkumyan, T. V. Priputnevich, et al.
767
ics in veterinary [10]. Further new recommendations were published by European Food Safety Authority (EFSA, 2005). Methodological regulations (MR) for sanitary and epidemiological estimation of safety and functional potential of probiotic microorganisms using for production of food products were developed in Russia [2]. In accordance to these MR, the sensitivity of lactobacillus strains to ABS should be evaluated by DDT or SDM of the antibiotic in liquid nutrient medium. These criteria were made for 16 substances from main groups of antibiotics used in medicine (penicillins, cephalosporins, carbapenems, aminoglycosides, tetracyclines, macrolides, lincosamines, glycopeptides, polymyxines, quinolones, and fluoroquinolones). Here we studied the sensitivity of most prevalent clinical strains of Lactobacillus (L. cripatus, L. iners, L. jensenii, and L. gasseri) isolated from vaginal secretion of pregnant women to ABS.
MATERIALS AND METHODS Antibiotic sensitivity of 123 strains of Lactobacillus was estimated: L. cripatus (21 strains), L. iners (68 strains), L. jensenii (16 strains), and L. gasseri (18 strains). Sensitivity was estimated by DDT as described elsewhere [2]. We used commercial disks with 12 antibiotics (ampicillin, cefazolin, cefotaxime, gentamycin, erythromycin, clindamycin, trimethoprim/sulfamethoxazole, tetracycline, ciprofloxacin, levofloxacin, vancomycin, and metronidazole; Bio-Rad).
Experiments were repeated 3 times, results were expressed as mean average. Results were analyzed in accordance to the criteria of estimation of lactobacillus sensitivity suggested in [2].
RESULTS All study strains were sensitive to ampicillin, cefazolin, cefotaxime, and vancomycin, and not resistant to metronidazole, trimethoprim/sulfamethoxazole, and levofloxacin (Table 1). Sensitivity to gentamycin, ciprofloxacin, clindamycin, tetracycline, and erythromycin varied depending on the type and strain of lactobacilli. All study strains of L. iners were resistant to tetracycline, but all other strains of lactobacilli were susceptible to this substance. Sensitivity to clindamycin was found in all strains of L. crispatus and L. jensenii, but all study strains of L. iners and L. gasseri were resistant to this ABS. All strains of L. crispatus and L. jensenii, but only 61.1% of L. gasseri cultures were resistant to gentamycin and ciprofloxacin. All strains of L. iners were sensitive to ciprofloxacin, but only 10.3% of them were sensitive to gentamycin. Sensitivity of lactobacilli to erythromycin also varied depending on the strain. The majority of strains resistant to ABS belonged to L. iners and L. gasseri (Fig. 1). We showed significant differences in the sensitivity of various strains of vaginal lactobacilli to ABS. Previous studies were performed on probiotic lactobacilli, which are the components of food or probiotics, or lactobacilli from microbiota of the gastrointestinal tract [1,4,8].
TABLE. Sensitivity of Various Types of Lactobacilli to ABS Lactobacillus spp. (n=123) ABS L. crispatus (n=21)
L. iners (n=68)
L. jensenii (n=16)
L. gasseri (n=18)
Ampicillin
S
S
S
S
Cefazolin
S
S
S
S
Cefotaxime
S
S
S
S
Ciprofloxacin
R
S
R
S/R
Levofloxacin
R
R
R
R
Clindamycin
S
R
S
R
Gentamycin
R
S/R
R
S/R
Erythromycin
S
S/R
S/R
S/R
Trimethoprim/sulfamethoxazole
R
R
R
R
Tetracycline
S
R
S
S
Vancomycin
S
S
S
S
Metronidazole
R
R
R
R
Note. R, resistant; S, susceptible; S/R, variable.
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Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 PHARMAСOLOGY AND TOXICOLOGY
Fig. 1. Frequency of isolation of lactobacillus types resistant to ABS prevalent in vaginal microbiota.
All lactobacillus strains isolated form vaginal secret are sensitive to ampicillin, cefazolin, cefotaxime, and vancomycin, and not resistant to metronidazole, trimethoprim/sulfamethoxazole, and levofloxacin. Various types of vaginal lactobacilli have variable sensitivity to gentamycin, clindamycin, erythromycin, ciprofloxacin, and tetracycline. Obtained data allow to conclude that therapy with metronidazole, levofloxacin, and trimethoprim/sulfamethoxazole does not affect the number of lactobacilli in the vagina. However treatment with penicillins, cephalosporins, and glycopeptides can promote a decrease in lactobacillus pool in the vagina. Variable sensitivity of certain strains should be taken into account during choosing of schemes of antibacterial treatment. For example, therapy with clindamycin can induce selective accumulation of L. iners and L. gasseri, and suppression or elimination of L. crispatus from vaginal microbiota. In accordance to the results of in vitro investigations, we made suggestions about the effects of therapy with antibiotics on the presence of various types of lactobacillus in vaginal microbiota, which should be further confirmed by clinical investigations. It is known [7] that L. iners is a lactobacillus, which quantitative level is firstly disposed in vaginal microbiota after antibiotic treatment. In accordance to several investigations [5,11] L. iners can be a marker of sensitivity to bacterial vaginosis, and L. crispatus is the prevalent type under normal conditions. Thus, the phenomenon of selective effects of ABS on lactobacillus composition of vaginal microbiota (L. iners remained in vaginal microbiota and has advantages of other types of lactobacilli, especially of L. crispatus) should be taken into account during treatment of bacterial vaginosis.
Obtained data on antibiotic sensitivity of clinical strains of lactobacilli show the possibility of restoration of their pool in vaginal microbiota during antibacterial therapy. This work was supported by the Ministry of Education and Science of the Russian Federation (State Contract No. 14.607.21.0019 from June 5, 2014).
REFERENCES 1. S. G. Botina, E. U. Poluektova, A. A. Glazova, et al., Mikrobiologiya, 80, No. 2, 1-9 (2011). 2. Methodological Regulations 2.3.2.2789-10 for Sanitary and Epidemiological Estimation of Safety and Functional Potential of Probiotic Microorganisms using for Production of Food Products [in Russian], Moscow (2010). 3. M. V. Sukhorukova, A. V. Timokhova, M. V. Eidel’shtein, and R. S. Kozlov, Klin. Mikrobiol. Antimikrob. Khimioter., 14, No. 3, 245-251 (2012). 4. M. R. D’Aimmo, M. Modesto, and B. Biavati, Int. J. Food Microbiol., 115, No. 1, 35-42 (2007). 5. T. Jakobsson, and U. Forsum, J. Clin. Microbiol., 45, No. 9, 3145 (2007). 6. J. H. Jorgensen, and J. F. Hindler, Clin. Infect. Dis., 44, No. 2, 280-286 (2007). 7. A. Kalra, C. T. Palcu, J. D. Sobel, and R. A. Akins, Curr. Infect. Dis. Rep., 9, No. 6, 485-500 (2007). 8. S. Mayrhofer, K. J. Domig, C. Mair, et al., Appl. Environ. Microbiol., 74, No. 12, 3745-3748 (2008). 9. V. Ocana, C. Silva, and M. E. Nader-Macias, Infect. Dis. Obstet. Gynecol., 2006, 118182 (2006). 10. Opinion of the Scientific Committee on Animal Nutrition on the Criteria for Assessing the Safety of Microorganisms Resistant to Antibiotics of Human Clinical and Veterinary Importance. Adopted on 3 July 2001, revised on 18 April 2002. 11. H. Verstraelen, R. Verhelst, G. Claeys, et al., BMC Microbiol., 9, 116 (2009).
766
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 PHARMAСOLOGY AND TOXICOLOGY
Effects of Antibiotic Treatment on the Lactobacillus Composition of Vaginal Microbiota A. R. Melkumyan, T. V. Priputnevich, A. S. Ankirskaya, V. V. Murav’eva, and L. A. Lubasovskaya Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 12, pp. 730-733, December, 2014 Original article submitted April 21, 2014 We analyzed sensitivity of 123 vaginal lactobacillus strains to antibacterial substances. All lactobacillus strains were sensitive to ampicillin, cefazolin, cefotaxime, and vancomycin, and insensitive to metronidazole, trimethoprim/sulfamethoxazole, and levofloxacin. Lactobacillus strains demonstrated different sensitivity to gentamycin, clindamycin, erythromycin, ciprofloxacin, and tetracycline. The phenomenon of preferential selective influence of antibacterial drugs on the composition of lactobacilli of the vaginal microbiota, in which some lactobacilli survive as part of the vaginal microbiota and have a selective advantage over other types of lactobacilli, should be taken into account during treatment of vaginal infections and dysbiosis. Key Words: vaginal microbiota; lactobacilli; L. crispatus; L. iners; antibiotic sensitivity Effects of antibacterial substances (ABS) on the resident component of vaginal microbiota during treatment of dysbiosis and inflammatory processes in the urogenital system have two points. Minimization of the effects of ABS on normal microflora can induce resistance of “useful” microflora to the used ABS. However, during long-term and frequent antibiotic therapy the normal microflora can obtain certain mechanisms of resistance to antibiotics and transfer resistance genes to pathogenic microorganisms and transitory microflora. The problem of administration of probiotics in parallel with antibiotic therapy is actively discussed in literature. Under these conditions, antibiotics affect not only pathogenic microorganisms, but also useful probiotic strains [1,3,4]. Standardization of the methods for estimation of phenotypic resistance of bacteria from normal microflora is a necessary condition for recognizing genotypes resistant to ABS. Current traditional standardized methods for estimation of the sensitivity of clinically significant bacteria can hardly be extrapolated to V. I. Kulakov Research Center for Obstetrics, Gynecology, and Perinatology, Ministry of Health of the Russian Federation, Moscow, Russia. Address for correspondence: alinamelkumyan@yandex. ru. A. R. Melkumyan
non-pathogenic bacteria, e.g., lactobacilli. Difficulties in evaluation of antibiotic sensitivity of Lactobacillus strains isolated from the blood of patients with endocarditis showed the necessity of development of standards for estimation of sensitivity of lactobacilli with consideration for their growth requirements and distribution in antibacterial medium. Several investigations for standardization of estimation of lactobacillus sensitivity were performed previously [1,4,6,9]. Various methodological approaches, nutrient media, regimens, and culturing terms and conditions were used in these experiments. Within the framework of EC ACEART project, standard operational procedures for estimation of the sensitivity of lactobacilli to antimicrobial substances using disk diffusion test (DDT), E-tests, and serial dilution method (SDM) in broth were developed. Experiments [8] showed that each method has advantages and disadvantages. “Agar” methods, e.g., E-test and DDT (especially E-test), have some advantages over SDM. Antibacterial resistance safety problems are certainly taken into account during development and marketing of probiotics. Resolution of the Scientific Committee on Animal Nutrition (SCAN; 2001) of Europe stated criteria for estimation of safety of microorganisms insensitive to antibiotics for the use as probiot-
0007-4888/15/15860766 © 2015 Springer Science+Business Media New York
A. R. Melkumyan, T. V. Priputnevich, et al.
767
ics in veterinary [10]. Further new recommendations were published by European Food Safety Authority (EFSA, 2005). Methodological regulations (MR) for sanitary and epidemiological estimation of safety and functional potential of probiotic microorganisms using for production of food products were developed in Russia [2]. In accordance to these MR, the sensitivity of lactobacillus strains to ABS should be evaluated by DDT or SDM of the antibiotic in liquid nutrient medium. These criteria were made for 16 substances from main groups of antibiotics used in medicine (penicillins, cephalosporins, carbapenems, aminoglycosides, tetracyclines, macrolides, lincosamines, glycopeptides, polymyxines, quinolones, and fluoroquinolones). Here we studied the sensitivity of most prevalent clinical strains of Lactobacillus (L. cripatus, L. iners, L. jensenii, and L. gasseri) isolated from vaginal secretion of pregnant women to ABS.
MATERIALS AND METHODS Antibiotic sensitivity of 123 strains of Lactobacillus was estimated: L. cripatus (21 strains), L. iners (68 strains), L. jensenii (16 strains), and L. gasseri (18 strains). Sensitivity was estimated by DDT as described elsewhere [2]. We used commercial disks with 12 antibiotics (ampicillin, cefazolin, cefotaxime, gentamycin, erythromycin, clindamycin, trimethoprim/sulfamethoxazole, tetracycline, ciprofloxacin, levofloxacin, vancomycin, and metronidazole; Bio-Rad).
Experiments were repeated 3 times, results were expressed as mean average. Results were analyzed in accordance to the criteria of estimation of lactobacillus sensitivity suggested in [2].
RESULTS All study strains were sensitive to ampicillin, cefazolin, cefotaxime, and vancomycin, and not resistant to metronidazole, trimethoprim/sulfamethoxazole, and levofloxacin (Table 1). Sensitivity to gentamycin, ciprofloxacin, clindamycin, tetracycline, and erythromycin varied depending on the type and strain of lactobacilli. All study strains of L. iners were resistant to tetracycline, but all other strains of lactobacilli were susceptible to this substance. Sensitivity to clindamycin was found in all strains of L. crispatus and L. jensenii, but all study strains of L. iners and L. gasseri were resistant to this ABS. All strains of L. crispatus and L. jensenii, but only 61.1% of L. gasseri cultures were resistant to gentamycin and ciprofloxacin. All strains of L. iners were sensitive to ciprofloxacin, but only 10.3% of them were sensitive to gentamycin. Sensitivity of lactobacilli to erythromycin also varied depending on the strain. The majority of strains resistant to ABS belonged to L. iners and L. gasseri (Fig. 1). We showed significant differences in the sensitivity of various strains of vaginal lactobacilli to ABS. Previous studies were performed on probiotic lactobacilli, which are the components of food or probiotics, or lactobacilli from microbiota of the gastrointestinal tract [1,4,8].
TABLE. Sensitivity of Various Types of Lactobacilli to ABS Lactobacillus spp. (n=123) ABS L. crispatus (n=21)
L. iners (n=68)
L. jensenii (n=16)
L. gasseri (n=18)
Ampicillin
S
S
S
S
Cefazolin
S
S
S
S
Cefotaxime
S
S
S
S
Ciprofloxacin
R
S
R
S/R
Levofloxacin
R
R
R
R
Clindamycin
S
R
S
R
Gentamycin
R
S/R
R
S/R
Erythromycin
S
S/R
S/R
S/R
Trimethoprim/sulfamethoxazole
R
R
R
R
Tetracycline
S
R
S
S
Vancomycin
S
S
S
S
Metronidazole
R
R
R
R
Note. R, resistant; S, susceptible; S/R, variable.
768
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 PHARMAСOLOGY AND TOXICOLOGY
Fig. 1. Frequency of isolation of lactobacillus types resistant to ABS prevalent in vaginal microbiota.
All lactobacillus strains isolated form vaginal secret are sensitive to ampicillin, cefazolin, cefotaxime, and vancomycin, and not resistant to metronidazole, trimethoprim/sulfamethoxazole, and levofloxacin. Various types of vaginal lactobacilli have variable sensitivity to gentamycin, clindamycin, erythromycin, ciprofloxacin, and tetracycline. Obtained data allow to conclude that therapy with metronidazole, levofloxacin, and trimethoprim/sulfamethoxazole does not affect the number of lactobacilli in the vagina. However treatment with penicillins, cephalosporins, and glycopeptides can promote a decrease in lactobacillus pool in the vagina. Variable sensitivity of certain strains should be taken into account during choosing of schemes of antibacterial treatment. For example, therapy with clindamycin can induce selective accumulation of L. iners and L. gasseri, and suppression or elimination of L. crispatus from vaginal microbiota. In accordance to the results of in vitro investigations, we made suggestions about the effects of therapy with antibiotics on the presence of various types of lactobacillus in vaginal microbiota, which should be further confirmed by clinical investigations. It is known [7] that L. iners is a lactobacillus, which quantitative level is firstly disposed in vaginal microbiota after antibiotic treatment. In accordance to several investigations [5,11] L. iners can be a marker of sensitivity to bacterial vaginosis, and L. crispatus is the prevalent type under normal conditions. Thus, the phenomenon of selective effects of ABS on lactobacillus composition of vaginal microbiota (L. iners remained in vaginal microbiota and has advantages of other types of lactobacilli, especially of L. crispatus) should be taken into account during treatment of bacterial vaginosis.
Obtained data on antibiotic sensitivity of clinical strains of lactobacilli show the possibility of restoration of their pool in vaginal microbiota during antibacterial therapy. This work was supported by the Ministry of Education and Science of the Russian Federation (State Contract No. 14.607.21.0019 from June 5, 2014).
REFERENCES 1. S. G. Botina, E. U. Poluektova, A. A. Glazova, et al., Mikrobiologiya, 80, No. 2, 1-9 (2011). 2. Methodological Regulations 2.3.2.2789-10 for Sanitary and Epidemiological Estimation of Safety and Functional Potential of Probiotic Microorganisms using for Production of Food Products [in Russian], Moscow (2010). 3. M. V. Sukhorukova, A. V. Timokhova, M. V. Eidel’shtein, and R. S. Kozlov, Klin. Mikrobiol. Antimikrob. Khimioter., 14, No. 3, 245-251 (2012). 4. M. R. D’Aimmo, M. Modesto, and B. Biavati, Int. J. Food Microbiol., 115, No. 1, 35-42 (2007). 5. T. Jakobsson, and U. Forsum, J. Clin. Microbiol., 45, No. 9, 3145 (2007). 6. J. H. Jorgensen, and J. F. Hindler, Clin. Infect. Dis., 44, No. 2, 280-286 (2007). 7. A. Kalra, C. T. Palcu, J. D. Sobel, and R. A. Akins, Curr. Infect. Dis. Rep., 9, No. 6, 485-500 (2007). 8. S. Mayrhofer, K. J. Domig, C. Mair, et al., Appl. Environ. Microbiol., 74, No. 12, 3745-3748 (2008). 9. V. Ocana, C. Silva, and M. E. Nader-Macias, Infect. Dis. Obstet. Gynecol., 2006, 118182 (2006). 10. Opinion of the Scientific Committee on Animal Nutrition on the Criteria for Assessing the Safety of Microorganisms Resistant to Antibiotics of Human Clinical and Veterinary Importance. Adopted on 3 July 2001, revised on 18 April 2002. 11. H. Verstraelen, R. Verhelst, G. Claeys, et al., BMC Microbiol., 9, 116 (2009).
