In vitro inhibition of commercial douche products against vaginal microflora.

Recently, vaginal douching has been associated with many health risks in women. The aim of this study was to analyze the effect of commercial douche products against various vaginal microorganisms, including lactobacilli. Seven commercial douches were tested against eight Lactobacillus clinical isolates and three type strains from the American Type Culture Collection. BV-associated bacteria included six strains of five genera: Gardnerella, Mobiluncus, Mycoplasma, Peptostreptococcus, and Ureaplasma. Two isolates of group B Streptococcus, and three species of Candida were also tested. The minimal inhibition concentrations and minimal contact times for these products against vaginal microorganisms were determined in broth cultures. Four antiseptic-containing douche products showed a strong inhibitory effect against all vaginal microorganisms tested with a short contact time (less than 1 min). Three vinegar-containing douche products selectively inhibited vaginal pathogens associated with bacterial vaginosis, group B streptococcal vaginitis, and candidiasis, but not lactobacilli. The antimicrobial effects of the commercial douche products varied among different brands and microbial species tested.

organisms from colonization or overgrowth, z Lactobacilli produce factors including lactic acid, hydrogen peroxide, and various bacteriocins, which may tend to exclude other microbes in the same environment. Therefore, factors that inhibit or eliminate lactobacilli may facilitate colonization or overgrowth of potentially pathogenic microorganisms in the vagina.
Vaginal douching has a long history and is practiced regularly by many American women and ado-lescent girls. [3][4][5][6] However, little is known about the risks versus personal benefits of this practice. Many clinical studies suggest a possible association between douching and various health risks in women. These include pelvic inflammatory disease, 7,s bacterial vaginosis (BV), [9][10][11] chlamydia, le and other sexually transmitted infections including HIV, 13-1s miscarriages, 16 premature birth of low birth-weight infants, 17 ectopic pregnancy, ls, 19 and cervical cancer. 2o Although most published data argue against douching, 7-2 two studies indicated that douching might be medically beneficial. 2-2e First, douching may significantly eliminate semen after sexual in-tercourse. This may decrease both the retention time and load of sexually transmitted pathogens in the vagina, el Second, by comparing douching and HIV prevalence in women, ez a study showed that douching with commercial antiseptic preparations is associated with a lower prevalence of HIV, but douching with noncommercial preparations is associated with a higher prevalence of HIV. Therefore, in addition to the flushing effect, the ingredients of douche solutions may also be critical.
Because douching may disrupt the balance of vaginal microbial ecology, its inhibitory effects on vaginal microorganisms, including the commensal lactobacilli, need to be studied. A prior in vitro study using only lactobacilli and antiseptic douche solutions showed that the douches might kill vaginal lactobacilli, z3 Another in vivo study tested more vaginal microorganisms, but it only analyzed two douche preparations, z4 Because the vaginal flora contains many different microorganisms in addition to lactobacilli, and because the composition of douche products varies from brand to brand, studies with more douche products and a more complete spectrum of vaginal microorganisms are needed to evaluate the association between douching and vaginal health. In the present study, therefore, we tested in vitro a range of vaginal microorganisms, including vaginal lactobacilli and pathogens associated with BV, vaginal candidiasis (VC), and group B streptococcal vaginitis, in response to seven douche products common to the U.S. market. Among these products, some contained different antiseptics, and some contained none.

MATERIALS AND METHODS
Seven vaginal douche products were purchased from local department stores or drug stores in the United States. Each product was arbitrarily assigned an alphabetical letter. The ingredients of these products as indicated on their boxes are presented in Table 1. Their pH values were measured and are also listed in Table 1. The ingredients of douche B were mixed immediately before use. The ingredients of douches F and G were similar; the difference was that douche G contained additional dyes and fragrance.
The vaginal microorganisms used in this study included a group of normal flora, lactobacilli, and three groups of pathogenic floras: BV-associated Additionally, two strains of group B streptococci (from Dr. Crag Rubens at University of Washington, Seattle) and three species of Candida were also included. A complete list of the microorganisms is shown in Table 2. Multiple species and strains of each group were used for the study in order to compare the range of their sensitivities to various douche products. The inhibitory effect of the douche products against vaginal microflora were determined by two previously described methods, z3 The first method measured the minimal inhibitory concentration (MIC) of each douche solution (after proper dilution) required to inactivate individual testing microorganisms. The   To measure MCT, actively growing bacterial or yeast cultures in mid-exponential phase were centrifuged at 5,000 rpm for 5 min and washed in phosphate-buffered saline (PBS). The PBS with pH 6.5 was used to wash lactobacilli, whereas the PBS with pH 7.3 was used to wash pathogens. The washed cells were resuspended in an undiluted douche solution at a final concentration of 106 cfu/ ml. The cells resuspended in PBS were used as controls. At different time intervals (0, 1, 5, 10, 15, 20, and 30 min), 0.5-ml aliquots of microorganismdouche suspension were removed, washed in PBS, and resuspended in appropriate broth medium. After anaerobic incubation at 37C for 24 h, the growth of each culture was determined with a spectrophotometer at OD6oo. For douches A to E, the MIC values against lactobacilli were relatively high, ranging from 25% to no effect. However, douches F and G exhibited much lower MIC values against the same testing organisms, ranging between 1.56% and 6.25%. Douches A and B (medicated with povidone-iodine at a final concentration of 0.3%) showed no significant effect on the Lactobacillus strains tested. Douches C and D showed no detectable effect against most microorganisms tested. The slight inhibitory effect of douche C on some vaginal Lactobacillus strains may be due also to its higher pH (7.2) as compared with the rest of products tested (pH range between 3.0 and 4.2). In general, the MIC values of these douche products against lactobacilli varied from strain to strain.

INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY I01
The MIC values of these douche solutions for BV-associated pathogens were lower than those for lactobacilli. The MIC values for douches A to E varied from 12.5% to 50%. The medicated douche B, designed for use by women with vaginitis symptoms, showed no significant differences from the effects of douches A, C, D, and E. Although douche C had a higher pH value, it was more effective against BV-associated pathogens than was the medicated douche B. Douches F and G exhibited significant antibacterial and antifungal activities against all microorganisms tested. The two G. vaginalis strains showed different sensitive patterns to these douche products tested. In general, the three yeast cultures were less sensitive to these douche products tested.
The MCT results are presented in Table 3. The contact time intervals were selected to simulate the actual presence of the douche solution in the va-gina. The douche products were tested at full strength without dilution. Douches B, C, F, and G required less than min to kill all of the tested Lactobacillus strains, BV-associated pathogens, group B streptococci, and 2 of 3 yeast cultures in vitro. No apparent effect on growth of lactobacilli, group B streptococci and U. urealyticum was observed after exposure to douches D and E for up to 30 min. Interestingly, douches D and E killed most BV-associated pathogens tested. They killed (7. vaginalis, P. tetradius, and Mycoplasma hominis upon contact for less than 1 min, and 31obiluncus curtisii upon contact for less than 5 min. These two douches also inhibited in vitro the growth of the three Candida species tested. Although douche A suppressed many lactobacilli after 5 to 30 min of exposure, it more effectively inhibited BVassociated pathogens (less than min) and group B streptococci (5 min). But it had virtually no effect on yeasts.

DISCUSSION
Recent clinical studies suggested that women who frequently douche are associated with an increased risk of pelvic inflammatory disease, 7,8 BV, 9-1 chlamydia, e and other sexually transmitted infections, including HIV, 13-1s miscarriages, 6 premature birth of low birth-weight infants, 7 ectopic pregnancy, [8][9][10][11][12][13][14][15][16][17][18][19] and cervical cancer, z Nonetheless, datae, ee that support douching exist. These include elimination of semen after sexual contact. As a result, it may decrease the retention time and load of sexually transmitted pathogens, presumably reducing the risk of contracting sexually transmitted diseases. Clearly, the effect of douching is controversial, and the mechanism is unknown. Several possibilities might have compromised the research results. First, in some women, douching may be in response to vaginal discomfort caused by preexisting infections. Second, douching may be associated with a high level of sexual activity, which by itself can be a risk factor for vaginal infection. 3  she already has an infection, a douche product containing antiseptics may conceivably help decrease the vaginal load of infecting bacteria. We also speculate that after the antimicrobial treatment, it may be helpful to apply a second product to facilitate the reestablishment of vaginal lactobacilli-perhaps a Lactobadllus vaginal suppository or acid gel.
In summary, this in vitro study suggested that the antimicrobial effects of the commercial douche products varied among different brands and microbial species tested. The antiseptic-containing douche products inhibited all microorganisms tested. The vinegar-containing products selectively inhibited pathogens associated with vaginal infections, but not vaginal lactobacilli.