Abstract
According to Russian classification, bacterial vaginosis (BV) and nonspecific vaginitis (NSV) are two different clinical conditions. The vaginal microbiota was evaluated in 200 women (50 healthy pregnant women (HPW) during the first trimester, 100 patients with BV and 50 patients with NSV). The samples received from both healthy women (121 samples) and patients (BV 241 samples, NSV 136 samples) contained lactobacilli (96.5%), peptostreptococci (52.2%), streptococci (36.4%), bifidobacteria (48.2%), and propionibacteria (34.3%). Other microorganisms (bacteroides, enterobacteria, yeasts, staphylococci) were recovered by culture in 1.2–27.8% of samples. The most common microorganisms found in the vaginal discharge samples of women with BV and NSV were opportunistic pathogens (staphylococci 32.3%, enterococci 17.5%, enterobacteria 28.2%, candida 21.3%, peptostreptococci 64.2%, peptococci 38.8%, gram-negative anaerobic bacteria 29.4%). In most cases, more than one species of microorganisms were present (simultaneously representatives of 1–3 (NSV) or 3–5 (BV) species). The vaginal lactobacillus content in healthy women was log 7–9 cfu/ml. The total number of vaginal lactobacilli in the samples from BV and NSV women was reduced by up to log 6–7 cfu/ml and log 3–4 cfu/ml, respectively; 41.9% of NSV samples contained < 103 cfu/ml of lactobacilli. All vaginal aerobic microorganisms isolated from the patients were resistant to 9 or more of 22 antibiotics tested. Microorganisms isolated were evaluated for their ability to restore nitrate and nitrite (Grees reagent) and to produce arginine decarboxylase (Moeller medium with arginine). Nitrate and nitrite reductases were produced by 25–28% of vaginal microbe strains isolated from HPW, 61–64% strains isolated from patients with NSV and 51–55% of those with BV (mostly belonging to enterobacteria, peptostreptococci, bacteroides, candida, and some others). Arginine decarboxylase activity was found in 41%, 53.4%, and 49.5% of strains isolated from these groups of women, respectively. The results obtained allow us to suppose that the determination of activity of enzymes investigated in vaginal strains or vaginal discharges may be used as additional diagnostic or screening tests for the predictive value of vaginal microbial ecology imbalance in BV and NSV.
Introduction
According to epidemiological studies, opportunistic infections of the female genital tract are increasing in prevalence and remain unresolved by therapy. Most of these infections are due to enterobacteria, bacteroides, corynobacteria, candida, and other facultative and anaerobic bacteria possessing potential pathogenicity Citation[1], Citation[2]. It is known Citation[1] that women with bacterial vaginosis (BV) or nonspecific vaginitis (NSV) represent a group at particular risk for fertility and pregnancy.
In Russia, opportunistic infections of the female genital tract were named nonspecific vaginitis (NSV). The functional condition of the female genital tract characterized by disorders of the vaginal microbiota without exact clinical symptoms that predisposed to the development of NSV led some Russian specialists to recommend the name bacterial vaginosis (BV). Some clinical and laboratory criteria and tests were proposed to differentiate between BV and NSV Citation[3], Citation[4]. Unfortunately, to date, the exact criteria for diagnosis of BV and NSV have been lacking.
It is known that nitric oxide (NO) can regulate regional blood flow, gut motility, water and electrolyte transport, and immunity. Some commensal gut strains are able to generate NO Citation[5], Citation[6]. We have not found data on the ability of vaginal bacterial strains to produce NO.
The aims of the present study were to evaluate the vaginal microbial ecosystem in healthy pregnant women (HPW) and nonpregnant patients with bacterial vaginosis (BV) and nonspecific vaginitis (NSV), to determine the susceptibility of various strains of opportunistic facultative aerobic bacteria isolated to several groups of common antimicrobial agents, and to determine the ability of various strains of vaginal bacteria to restore nitrate and nitrite and production of arginine decarboxylase in vitro.
Materials and methods
Microorganisms
The study population consisted of 200 women (25–40 years old), 50 of whom were HPW (first trimester) and 150 were nonpregnant women (50 with NSV, 100 with BV). Diagnosis of NSV and BV was based on the laboratory indexes (cytological, microbiological, and immunological data) Citation[3]. All women were enrolled in the study between 2000 and 2003.
Microbiological investigations were performed in all women at the first check-up and 14–21 days later. A total of 498 vaginal specimens were obtained by rolling a sterile cotton swab across the vaginal wall. Then every specimen was placed into 2 ml of a prereduced sterile balanced salt solution and 100 µl of sample was inoculated onto different solid media. Standard media (Columbia Agar Base, Shaedler Agar, Endo Agar Base, Mannitol Salt Agar, Bile Esculin Azide Agar, MRS Agar, TSN Agar, Sabouraud Medium (Pronadisa, Hispanlab, Spain; Difco Laboratories, USA), Bifidus Medium (Obolensk, Moscow Region, Russia) allowing growth of facultative and/or anaerobic bacteria and candida and appropriate techniques were employed Citation[7–9]. When necessary, some media were enriched with sheep blood, egg yolk or antimicrobial agents. Commercial gas generating systems were also available (GENbox anaer, bioMerieux, France). The 2788 bacterial and candida strains isolated were identified to genus and species level on the basis of Gram's stain and cultural and biochemical tests with API systems (bioMerieux) or Mikro-La-Test (PLIVA-Lachema Diagnostika, Brno, CZ).
Measurement of the sensitivity of facultative anaerobic bacteria isolated to 22 antimicrobial agents (β-lactams, aminoglycosides, macrolides, quinolones, glycopeptides, chloramphenicol, tetracyclines, and others) was performed by disc diffusion methods on meat-peptone agar with or without the addition of blood Citation[7].
Bacterial nitrate (nitrite) reductase production was determined by using qualitative reaction with Grees reagents. Bacterial arginine decarboxylase activity was determined by qualitative reaction using Moeller medium (Difco) with 1% L-arginine hydrochloride Citation[9]. The inoculation dose of bacteria was 108 cfu. Results were evaluated after anaerobic incubation at 37°C for 48–96 h.
Results and Conclusions
All data presented in the text and tables have been calculated on the basis of specimen numbers taken from healthy pregnant women and sick women at day 0 and 14–21 days (one and more often two or three vaginal swabs were taken from each woman and were investigated microbiologically). Thus, 121 vaginal specimens taken from 50 HPW and 377 specimens taken from 150 women with vaginal infection were investigated. As a result 693 and 2095 microbial strains were isolated from healthy women and patients, respectively.
The frequency of recovery of vaginal microorganisms from women with BV and NSV was compared with that of healthy women ().
Table I.  Vaginal flora of healthy pregnant women (HPW), patients with bacterial vaginosis (BV), and nonspecific vaginitis (NSV).
This study shows that the spectrum and number of vaginal microorganisms had essential differences in all groups investigated. The samples received from both healthy women and patients contained lactobacilli (96.5%), peptostreptococci (52.2%), streptococci (36.4%), bifidobacteria (48.2%), and propionibacteria (34.3%). Other microorganisms (bacteroides, enterobacteria, yeasts, staphylococci) were recovered by culture in 1.2–27.8% of samples. The most common microorganisms found in the vaginal discharge samples of women with BV and NSV were opportunistic pathogens (staphylococci 32.3%, enterococci 17.5%, enterobacteria 28.2%, candida 21.3%, peptostreptococci 64.2%, peptococci 38.8%, gram-negative anaerobic bacteria 29.4%). In most cases, more than one species of microorganisms were present (simultaneously representatives of 1–3 (NSV) or 3–5 (BV) species). Opportunistic microorganisms isolated in NSV patients were often found in monoculture (>106 cfu/ml).
The vaginal lactobacillus content in healthy women was log 7–9 cfu/ml. The total number of vaginal lactobacilli in the samples from BV and NSV women was reduced by up to log 6–7 cfu/ml and log 3–4 cfu/ml, respectively. Lactobacilli could be isolated in the NSV patients in only a few cases; 41.9% of samples in the NSV group contained lactobacilli in numbers <103 cfu/ml. So our results confirmed the data of other investigators Citation[1] that increase of opportunistic pathogens in the female genital tract always correlates with decreased frequency of lactobacilli isolation.
Opportunistic facultative aerobic pathogens isolated from women with BV and NSV were more resistant to chemotherapeutic agents than bacteria isolated from vaginas of HPW (). All vaginal aerobic microorganisms isolated from the patients were resistant to 9–12 of the 22 antimicrobial agents investigated. This difference may be explained by more frequent antibiotic use by sick women compared with healthy women.
Table II.  Isolation frequency of vaginal facultative anaerobic multiresistant bacterial strains in healthy pregnant women (HPW) and patients with bacterial vaginosis (BV) and nonspecific vaginitis (NSV).
Data of experimental investigations connected with detection frequency of vaginal specimens and vaginal strains with positive nitrate reductase, nitrite reductase, and arginine decarboxylase activities are presented in and .
Table III.  Detection frequency of vaginal specimens with positive nitrate reductase, nitrite reductase, and arginine decarboxylase activities.
Table IV.  Detection frequency of vaginal strains with positive nitrate reductase, nitrite reductase, and arginine decarboxylase activities.
According to data presented in , simultaneous positive nitrate reductase, nitrite reductase, and arginine decarboxylase activities were observed in 4.9% of vaginal samples received from HPW, in 8.7% from patients with BV, and in 19.8% from patients with NSV. In all, 3.2% of HPW investigated had vaginal strains that were able to restore simultaneous nitrate and nitrite and produced arginine decarboxylase in anaerobic conditions in vitro. Such strains could be isolated in 21.3% of patients with NSV and 17.2% of those with BV. Among 318 lactobacillus strains investigated (HPW 124, BV 110, NSV 84 strains), 14–16% of bacterial cultures were able to produce nitrate reductase, nitrite reductase, and/or arginine decarboxylase. The number of enzyme-forming lactobacillus strains was approximately equal in all groups observed.
So our results enabled us to find a direct correlation between the condition of the vaginal microbial ecology of adult women and isolation frequency of strains possessing enzymes associated with NO production. Determination of nitrate (nitrite) reductase and arginine decarboxylase activities in the whole vaginal specimens taken or in the vaginal microbial strains isolated using simple qualitative methods (in anaerobic conditions) may be recommended as additional diagnostic or screening tests for the predictive value of vaginal microbial ecology imbalance in BV and NSV Citation[10]. It is necessary to carry out additional investigations to understand the role of the microbial production or inactivation of NO in fertility or pregnancy problems.
References
- Larsen B. Vaginal flora in health and disease. Clin Obstet Gynecol 1993; 36: 107–21
- Suhyna MA, Saveljev VN. Epidemiology and pathogenicity of clinically important anaerobes. Epidemiology and Vaccine Prophylactica Moscow 2005;1:37–41 [ in Russian].
- Method of vaginal microbiota estimation. Patent RU 2249821 C1. 27. 04. 2004.
- Onischenko GG, Aleshkin VA, Afanasyev SS, Pospelova VV, eds. Immunobiological medicines and perspective of their using in the infectology. Moscow, 2002 [ in Russian].
- Sobko T. Influence of the microflora on gastrointestinal nitric oxide generation. Stockholm, Layout Ringvor Hagglof, 2006.
- Ivashkin TI, Drapkina OM. Clinical importance of nitric oxide and heat shock proteins. Moscow: Geotar-Med, 2001:88 [in Russian].
- Menshikov BB, ed. Clinical laboratory analitica. v. IV. Clinical microbiology. Moscow: Agat-Med, 2003 [in Russian].
- Bollows A, Hausler WJ, Hermann KL, Isenberg HD, Shadomy HJ, eds. Manual of clinical microbiology, 5th edn. Washington, DC: American Society for Microbiology, 1991:1364.
- Difco manual. Dehydrated culture, media and reagents for microbiology, 10th edn. Detroit, MI: Difco, 1994.
- Aleshkin VA, Shenderov BA, Voropaeva EA. Vagina microbial ecology in healthy women and patients with vaginosis (BA) and nonspecific vaginitis (NSV). Abstracts of the 3rd World Congress on Anaerobic Bacteria and Infections. Glasgow, UK, 7–9 May, 2003:17.