Effect of transport time, temperature, and concentration on the survival of group B streptococci in amies transport medium.

The impact of transport time and temperature on survival of group B streptococci (GBS) in Amies transport medium was evaluated. Viability of 10 or more CFU of GBS was maintained for 4 days at 24 or 3 degrees C. However, there was a significant decrease in viability for GBS held at 30 degrees C for 4 days.

Safety and efficacy evaluations for vaginal and rectal use of BufferGel in the macaque model.

BACKGROUND: The nonhuman primate model allows for safety and efficacy testing of topical microbicide products. GOAL: The goal of this study was to evaluate the safety and efficacy of vaginal and rectal applications of BufferGel (ReProtect, Inc.). STUDY DESIGN: The safety of repeated product applications was evaluated by microflora, pH, vaginal colposcopy, and rectal lavage. To test efficacy in preventing chlamydia, infection was documented by culture and nucleic acid amplification tests. RESULTS: Repeated vaginal or rectal applications of BufferGel were not associated with significant changes in microflora. BufferGel use had a transient acidifying effect on vaginal and rectal pH. Colposcopic observations remained relatively normal in all test animals. A slightly increased incidence of epithelial desquamation was noted after rectal product use compared with the control group. BufferGel did not prevent cervical or rectal chlamydial infection. CONCLUSION: BufferGel has an acceptable safety profile after repeated vaginal and rectal use, but does not prevent chlamydial infection in the macaque models.

Optimization of media for detection of hydrogen peroxide production by Lactobacillus species.

A healthy vaginal ecosystem has been shown to be protective against the acquisition of human immunodeficiency virus and gonorrhea, and women who are colonized with H(2)O(2)-producing lactobacilli are more likely to maintain a normal vaginal flora than women with lactobacilli that do not produce H(2)O(2). The purpose of this study was to formulate a testing medium that better supports the growth and detection of H(2)O(2) by a broader range of lactobacilli than a published, widely used agar formulation (TMB). The new medium (TMB-Plus) consists of brucella agar base, 3,3',5,5'-tetramethylbenzidine, horseradish peroxidase, starch, vitamin K, hemin, magnesium sulfate, manganese sulfate, and horse serum. To validate the new formula, 256 vaginal isolates and ATCC strains were inoculated onto TMB-Plus and, for comparison, onto TMB. Growth was enhanced for 69% of the isolates on TMB-Plus, and 48% had enhanced color production. The percentage of H(2)O(2)-positive isolates increased from 71% on TMB to 79% on TMB-Plus. Formulations using Rogosa or MRS agar base in combination with peroxidase and a chromogen did not support the growth of all of the strains of Lactobacillus, and fewer H(2)O(2)-producing strains were detected on these formulations than on TMB-Plus. This new medium better supports the growth of a wider range of Lactobacillus strains isolated from the vagina and enhances the color production of H(2)O(2)-producing strains.

An association of cervical inflammation with high-grade cervical neoplasia in women infected with oncogenic human papillomavirus (HPV).

Previous reports of genital conditions, such as nonspecific genital infection/sore or vaginal discharge associated with cervical cancer (L. A. Brinton et al., J. Natl. Cancer Inst. (Bethesda), 79: 23-30, 1987; C. J. Jones et al., Cancer Res., 50: 3657-3662, 1990), suggest a possible link between either genital tract inflammation or changes in bacteria flora consistent with bacterial vaginosis (BV) and cervical cancer. To test whether changes in vaginal bacterial flora or the degree of cervical inflammation are associated with women having a human papillomavirus (HPV) infection or with women infected with oncogenic HPV having high-grade cervical lesions (high-grade squamous intraepithelial lesions or cancer), we conducted a case-control study of women <50 years old enrolled in the Costa Rican natural history study of HPV and cervical neoplasia. To test whether BV and inflammation were associated with HPV DNA positivity, Analysis 1 was restricted to women with no or mild (low-grade or equivocal) cytological abnormalities, and the degree of inflammation and Nugent score (a measure of BV) were compared between women infected (n = 220) and not infected (n = 130) with HPV. To test whether BV and inflammation were associated with high-grade lesions, Analysis 2 was restricted to women infected with oncogenic HPV, and the degree of inflammation and Nugent score were compared between women with (n = 95) and without (n = 158) high-grade cervical lesions. In Analysis 1, BV and cervical inflammation were not associated with HPV infection. In Analysis 2, BV was not associated with high-grade lesions. However, we found a marginally significant positive trend of increasing cervical inflammation associated with high-grade lesions in oncogenic HPV-infected women, (P(trend) = 0.05). Overt cervicitis was associated with a 1.9-fold increase in risk of high-grade lesions (95% confidence interval, 0.90-4.1). The results of this study suggest that cervical inflammation may be associated with high-grade lesions and may be a cofactor for high-grade cervical lesions in women infected with oncogenic HPV.

The pig-tailed macaque rectal model: microflora and chlamydial infection.

BACKGROUND: A topical microbicide should protect against acquisition of sexually transmitted infection during both vaginal and rectal intercourse. The rectal microflora of the Macaca nemestrina (pig-tailed macaque) and humans were examined, as well as the histopathology of rectal tissues. In a subset of macaques, a human rectal isolate of Chlamydia trachomatis was inoculated into the rectum to establish rectal chlamydial infection. GOAL: To evaluate the comparability of the pig-tailed macaque rectal model with humans. STUDY DESIGN: Rectal swabs were collected for microbiologic analysis to characterize normal microflora in pig-tailed macaques and humans. Subsequently, 10 macaques received a rectal inoculation with C trachomatis, serovar D, prepared from a clinical rectal isolate. RESULTS: The rectal microflora of pig-tailed macaques (n = 80) were found to be comparable with the rectal flora of humans (n = 40). The prevalence of Lactobacillus in the rectum was higher in the macaques than in humans. Coliform and Enterococcus were decreased in the macaques, as compared with those of humans. In 9 of 10 macaques, rectal chlamydial infection was confirmed by culture or ligase chain reaction on days 2, 7, and 14 after inoculation. The test results were positive for rectal chlamydial infection by ligase chain reaction only for the remaining animal on day 14 after inoculation. CONCLUSIONS: The findings demonstrate that the rectal environment of the pig-tailed macaque is a useful model for further evaluation of newly developed topical microbicides for rectal use. Furthermore, such products can be evaluated for protection against rectal chlamydial infection in this model.

Effect of chlorhexidine on genital microflora, Neisseria gonorrhoeae, and Trichomonas vaginalis in vitro.

BACKGROUND: Chlorhexidine is a disinfectant that has been used in skin and mouth washes and as a preservative in some vaginal lubricants. A gel containing 0.25% chlorhexidine gluconate has been found to be effective against Chlamydia trachomatis in vitro and in animal models. Applied vaginally, 5 g of this gel could achieve vaginal fluid concentrations of < or = 1250 microg/ml. GOAL: To test the in vitro activity of chlorhexidine in a gel over a pH range of 4 to 8 in the presence or absence of blood. STUDY DESIGN: Organisms were exposed to chlorhexidine for 30 minutes to 2 hours, and the minimum cidal concentration (MCC) was calculated. RESULTS: The MCC for Neisseria gonorrhoeae was 25 microg/ml at 30 minutes and 12.5 microg/ml at 1 to 2 hours of exposure, whereas the MCC for Trichomonas vaginalis was 1250 microg/ml. Chlorhexidine was more active at pH 8 than pH 4, and less active in the presence of blood. The MCC for Lactobacillus crispatus was 1250 microg/ml at pH 4 and only 125 microg/ml at pH 8. CONCLUSIONS: Based on its in vitro activity, chlorhexidine may be an appropriate topical microbicide for prevention of gonorrhea, but not for prevention of trichomoniasis. This study suggests that the presence of blood and pH affect the activity of chlorhexidine against genital pathogens and commensals.

The effects of three nonoxynol-9 preparations on vaginal flora and epithelium.

To evaluate the effects of nonoxynol-9 (N-9) on the vaginal flora and epithelium, 48 women (16 in each group) were evaluated by use of quantitative vaginal cultures and colposcopy. at baseline and at 0.5, 4, 24, 48, and 72 h after insertion of one of three N-9 preparations (4% gel [Conceptrol], 3.5% gel [Advantage-24], or a 28% vaginal contraceptive film). The proportion positive for H2O2+ or H2O2- lactobacilli did not change significantly with any of the preparations, but lactobacilli concentrations decreased transiently. Both the proportion of women with Gardnerella vaginalis and the concentration of G. vaginalis decreased transiently. The proportion of women with Escherichia coli increased with the 4% gel, and the concentration increased with all preparations. The number with anaerobic gram-negative rods increased, although the concentrations decreased. Symptoms and colposcopic abnormalities were rare. Changes in levels of vaginal bacteria were transient after single applications of N-9, but adverse effects may be enhanced with frequent, chronic use.

Effects of multiple applications of benzalkonium chloride and nonoxynol 9 on the vaginal epithelium in the pigtailed macaque (Macaca nemestrina).

OBJECTIVE: Safe and effective vaginally applied microbicides could help to control the continuing spread of sexually transmitted diseases. STUDY DESIGN: This study used nonhuman primates to test the effects of multiple applications of nonoxynol 9, benzalkonium chloride, or a combination on vaginal flora and lower reproductive tract tissues. Fourteen monkeys (Macaca nemestrina) received daily vaginal applications of nonoxynol 9, benzalkonium chloride, or both for 3 to 4 days. Vaginal microflora and colposcopic observations were made at baseline and during and after completion of treatments. Cervical biopsy specimens were collected from a subset of animals. RESULTS: Cervical erythema and vaginal erythema were observed in all 3 treatment groups. Cervical papillae and epithelial disruption were present in both the nonoxynol 9 and the nonoxynol 9 plus benzalkonium chloride groups. Vaginal epithelial disruption was noted in both the benzalkonium chloride and the nonoxynol 9 plus benzalkonium chloride groups. Cervical biopsy specimens from each group revealed acute inflammatory infiltrates with occasional plasma cells and lymphoid follicles. Detection of most microorganisms, including viridans streptococci, decreased in the benzalkonium chloride and the nonoxynol 9 plus benzalkonium chloride groups. Detection of Lactobacillus species decreased in the benzalkonium chloride group. All microflora levels recovered after several days without microbicide use. CONCLUSIONS: Although nonoxynol 9 is currently the only microbicide approved for use as a spermicide in the United States, its repeated use may be detrimental to the epithelial tissues of the female reproductive tract. Benzalkonium chloride, currently approved for use in other countries, not only may damage epithelial tissues but also appears to reduce the population of potentially protective Lactobacillus species in the vagina.

Race/ethnicity, vaginal flora patterns, and pH during pregnancy.

OBJECTIVES: To investigate the relationship between bacterial vaginosis during pregnancy and black race/ethnicity. STUDY DESIGN: Gram staining was used to evaluate vaginal flora in 842 women at 24 to 29 weeks' gestation. RESULTS: Overall, 22.3% of blacks and 8.5% of whites had bacterial vaginosis. Vaginal pH and flora differed significantly by race/ethnicity; blacks were more likely to have pH > or = 4.5, no lactobacilli, small gram-variable and -negative rods, and Mobiluncus compared with whites (odds ratios 1.6, 1.5, 1.4, and 10.6, respectively). Quantity of morphotypes also differed, especially for Mobiluncus. Among women with Mobiluncus present (12.0% of blacks and 1.3% of whites), 73.3% of blacks compared with 40.0% of whites had the highest level. Adjustment for sociodemographics, sexual activity, sexually transmitted diseases, health behavior, and sexual hygiene did not explain these differences. CONCLUSION: We observed race/ethnicity differences in vaginal flora ecology. These differences may ultimately play a role in the larger proportion of preterm deliveries among black women compared with white women.

0.25% chlorhexidine gluconate gel. A protective topical microbicide.

BACKGROUND AND OBJECTIVES: An estimated 4 million new cases of chlamydial infection occur each year. This experiment assessed the effects of a vaginally applied gel formulation of 0.25% chlorhexidine gluconate on chlamydial infection and on the vaginal ecosystem. STUDY DESIGN: Twelve monkeys were treated with a single application of 0.25% chlorhexidine gluconate. These animals were assessed for changes in vaginal flora before and at 30 minutes, 1 day, and 2 days postapplication by microbiologic analysis. Cervical and vaginal tissues were assessed by colposcopy at each time point. Five monkeys received a single application of 0.25% chlorhexidine gluconate gel followed (30 minutes) by a cervical inoculation with Chlamydia trachomatis. Four monkeys were inoculated with Chlamydia only. Cervicovaginal tissues were assessed via modified colposcopy, vaginal swabs were collected for assessment of vaginal flora, and cervical swabs were collected for detection of Chlamydia (culture/ligase chain reaction) at baseline and days 1, 2, and 7 postinoculation. RESULTS: Changes in vaginal flora were minimal in all monkeys. Application of 0.25% chlorhexidine gluconate did not affect adversely vaginal colonization by lactobacilli. All chlamydial infection control monkeys were infected, whereas none of the five monkeys pretreated with chlorhexidine gluconate were positive for C. trachomatis by culture or ligase chain reaction. Colposcopic observations remained largely unchanged in all groups. CONCLUSIONS: A 0.25% chlorhexidine gluconate gel was protective against chlamydial infection in all animals tested, had no adverse effect on the vaginal flora, and had minimal effect on cervicovaginal tissues after a single application.

HIV-1 infection associated with abnormal vaginal flora morphology and bacterial vaginosis.

BACKGROUND: In-vitro research has suggested that bacterial vaginosis may increase the survival of HIV-1 in the genital tract. Therefore, we investigated the association of HIV-1 infection with vaginal flora abnormalities, including bacterial vaginosis and depletion of lactobacilli, after adjustment for sexual activity and the presence of other sexually transmitted diseases (STDs). METHODS: During the initial survey round of our community-based trial of STD control for HIV-1 prevention in rural Rakai District, southwestern Uganda, we selected 4718 women aged 15-59 years. They provided interview information, blood for HIV-1 and syphilis serology, urine for detection of Chlamydia trachomatis and Neisseria gonorrhoeae, and two self-administered vaginal swabs for culture of Trichomonas vaginalis and gram-stain detection of vaginal flora, classified by standardised, quantitative, morphological scoring. Scores 0-3 were normal vaginal flora (predominant lactobacilli). Higher scores suggested replacement of lactobacilli by gram-negative, anaerobic microorganisms (4-6 intermediate; 7-8 and 9-10 moderate and severe bacterial vaginosis). FINDINGS: HIV-1 frequency was 14.2% among women with normal vaginal flora and 26.7% among those with severe bacterial vaginosis (p < 0.0001). We found an association between bacterial vaginosis and increased HIV-1 infection among younger women, but not among women older than 40 years; the association could not be explained by differences in sexual activity or concurrent infection with other STDs. The frequency of bacterial vaginosis was similar among HIV-1-infected women with symptoms (55.0%) and without symptoms (55.7%). The adjusted odds ratio of HIV-1 infection associated with any vaginal flora abnormality (scores 4-10) was 1.52 (95% CI 1.22-1.90), for moderate bacterial vaginosis (scores 7-8) it was 1.50 (1.18-1.89), and for severe bacterial vaginosis (scores 9-10) it was 2.08 (1.48-2.94). INTERPRETATION: This cross-sectional study cannot show whether disturbed vaginal flora increases susceptibility to HIV-1 infection. Nevertheless, the increased frequency of HIV-1 associated with abnormal flora among younger women, for whom HIV-1 acquisition is likely to be recent, but not among older women, in whom HIV-1 is likely to have been acquired earlier, suggests that loss of lactobacilli or presence of bacterial vaginosis may increase susceptibility to HIV-1 acquisition. If this inference is correct, control of bacterial vaginosis could reduce HIV-1 transmission.

PIP: A study of 4718 women 15-49 years old in southwestern Uganda's Rakai district suggests that the loss of lactobacilli and the presence of bacterial vaginosis may increase susceptibility to HIV-1. These women were part of an ongoing community-based trial of sexually transmitted disease (STD) control as a strategy for AIDS prevention. The rate of HIV-1 infection among these women was 19.5%. Moderate bacterial vaginosis was detected in 44.5% of women, while 6.4% had severe bacterial vaginosis. STD rates were 10.2% for active or recent syphilis, 22.4% for trichomonas, 2.4% for gonorrhea, and 3.6% for chlamydia. The HIV rate was lowest (14.2%) among women with normal bacterial flora and highest (26.7%) among those with severe bacterial vaginosis. The HIV-abnormal flora association was higher in younger women, for whom HIV acquisition is likely to be recent, than in older women. In multiple logistic regression analyses adjusted for age, number of sexual partners, trichomonas, and syphilis, the odds ratio (OR) for HIV-1 infection associated with any abnormal vaginal flora was 1.52 (95% confidence interval [CI], 1.22-1.90) and with all bacterial vaginosis was 1.56 (95% CI, 1.24-1.97). These adjusted ORs were 1.50 (95% CI, 1.18-1.89) for moderate and 2.08 (95% CI, 1.48-2.94) for severe bacterial vaginosis. A cross-sectional study cannot ascertain whether the abnormalities in vaginal flora occurred before or after HIV seroconversion. However, the significantly increased proportion of HIV-infected women with depleted or absent vaginal lactobacilli is consistent with in vitro studies showing that hydrogen peroxidase-producing lactobacilli in vaginal flora inhibit HIV-1 viral replication. If the ongoing prospective study suggests a causal association with HIV, treatment of abnormal flora or bacterial vaginosis with inexpensive, effective drugs such as metronidazole may be indicated.

Vaginal colonization by Escherichia coli as a risk factor for very low birth weight delivery and other perinatal complications.

This study evaluated the relationship of vaginal Escherichia coli colonization to birth weight <1500 g and other perinatal complications in a cross-sectional study of 2646 women at the University of Washington Medical Center, Seattle, between October 1992 and January 1995. Vaginal E. coli colonization was more strongly associated with delivery at <34 weeks (relative risk [RR], 1.7; 95% confidence interval [CI], 1.3-2.3) and very low birth weight (RR, 1.9; 95% CI, 1.3-2.7) than with prematurity between 34 and 36 weeks or low birth weight. Heavy growth of E. coli had a higher risk of very low birth weight than light growth (RR, 2.4; 95% CI, 1.0-6.2). It may be important to screen and treat pregnant women for genital tract colonization with E. coli during prenatal care.

Effects of nonoxynol-9 on vaginal microflora and chlamydial infection in a monkey model.

BACKGROUND AND OBJECTIVES: Nonoxynol-9, an intravaginal microbicide, is chlamydiacidal in vitro but also cytotoxic. This study examines the effects of nonoxynol-9 in vivo, using a pigtail macaque model of chlamydial cervicitis. GOALS: To establish a minimum infectious dose of Chlamydia trachomatis in the macaque, and to observe the effects of a single dose of nonoxynol-9 on efficiency of chlamydial infection, vaginal microflora, and cervicovaginal irritation. STUDY DESIGN: The effects of 4% nonoxynol-9, C. trachomatis (5,000 or 10,000 IFU) or both nonoxynol-9 application and chlamydial infection were studied in 17 macaques. RESULTS: Following a single application of nonoxynol-9, chlamydial infection was prevented in 4 of 6 monkeys infected with 10,000 IFU; there was a transient decrease in anaerobic gram-negative rods (P < 0.05) and Peptostreptococci (P > 0.05), but no change in Lactobacillus. Mild cervicovaginal irritation was observed in the monkeys. CONCLUSIONS: A single dose of nonoxynol-9 causes minimal vaginal flora and epithelial irritation, and may be useful for prevention of chlamydial infection.

The vaginal microflora of pig-tailed macaques and the effects of chlorhexidine and benzalkonium on this ecosystem.

BACKGROUND AND OBJECTIVES: To characterize normal vaginal microflora of pig-tailed macaques and to evaluate two commonly used intravaginal compounds, chlorhexidine (CHG), a vaginal antiseptic (Surgilube, E. Fougera, Melville, NY), and benzalkonium chloride (BZK) (spermicidal contraceptive) in this monkey model to assess effects on the vaginal microflora. STUDY DESIGN: Vaginal swabs were collected for microbiologic analysis to characterize normal flora. Subsequently, the vagina was exposed to either CHG or BZK twice at 24-hour intervals. RESULTS: The vaginal microflora of 26 pig-tailed macaques was found to be remarkably similar to the vaginal flora of the human with respect to frequency of vaginal colonization by H2O2-producing lactobacilli, Prevotella species, and several other microorganisms. After two vaginal applications at 24-hour intervals, CHG had only small effects on the vaginal microflora of five animals. By contrast, BZK applied by the same protocol had profound adverse effects on the lactobacilli and Peptostreptococcus and more transient effects on vaginal Prevotella and viridans streptococci of six animals. CONCLUSIONS: These observations demonstrate that the vaginal microflora of the pig-tailed macaque is a useful model in which to further evaluate newly developed intravaginal contraceptives that may be microbicidal and/or virucidal before widespread intravaginal use in women.

The normal vaginal flora, H2O2-producing lactobacilli, and bacterial vaginosis in pregnant women.

In this study of the vaginal flora of 171 pregnant women in labor at term, the flora was categorized as normal (Lactobacillus predominant), intermediate, or representative of bacterial vaginosis (BV) on the basis of a vaginal smear. BV was diagnosed in 39 women (23%); the vaginal flora was classified as normal in 50% of cases and as intermediate in 27%. H2O2-producing lactobacilli were recovered from 5% of women with BV, 37% of those with an intermediate flora, and 61% of those with a normal flora. H2O2-negative lactobacilli were equally frequent (57%-65%) in all three groups. The microorganisms most frequently recovered from women with BV included Gardnerella vaginalis, Prevotella bivia/disiens, Bacteroides ureolyticus, Prevotella corporis/Bacteroides levii, Fusobacterium nucleatum, Mobiluncus species, Peptostreptococcus prevotii, Peptostreptococcus tetradius, Peptostreptococcus anaerobius, viridans streptococci, Ureaplasma urealyticum, and Mycoplasma hominis (P < .05 for each). The presence of all but three of these organisms was inversely related to vaginal colonization by H2O2-producing lactobacilli; the exceptions were B. ureolyticus, F. nucleatum, and P. prevotii. Other microorganisms were equally frequent among women with and without BV. We conclude that specific groups of anaerobes are associated with BV in this population and that a strong association exists between species associated with BV and those inhibited by H2O2-producing lactobacilli.

Rapid presumptive identification of black-pigmented gram-negative anaerobic bacteria by using 4-methylumbelliferone derivatives.

A rapid method for presumptive identification of black-pigmented gram-negative anaerobic rods was developed. Using filter paper spot tests for indole production, sialidase, alpha-glucosidase, beta-glucosidase, alpha-fucosidase, and trypsinlike enzyme activities, 100% of Porphyromonas gingivalis, Prevotella intermedia, and Bacteroides levii and 89% of Prevotella corporis isolates were correctly identified to the species level. Porphyromonas asaccharolytica and Porphyromonas endodontalis could not be differentiated from each other but could be distinguished from all other species tested. Similarly, Prevotella denticola, Prevotella loescheii, and Prevotella melaninogenica could not be differentiated from each other. The methods described are based on 4-methylumbelliferone derivatives of the various substrates and are simple to perform, rapid (less than 15 min), and applicable to difficult-to-cultivate anaerobic rods.

Vaginal Bacteroides species are associated with an increased rate of preterm delivery among women in preterm labor.

The relationship of high concentrations of vaginal microorganisms with an increased rate of preterm delivery (less than or equal to 34 weeks) among women in preterm labor was evaluated. Quantitative vaginal cultures were obtained from 211 women in preterm labor between gestational ages of 22 and 34 completed weeks. Preterm delivery occurred in 139 (66%). The rate of preterm delivery was determined by Cox proportional hazards models. Women with greater than 10(7) cfu/ml of vaginal fluid of facultative lactobacilli had a 40% decreased rate of preterm delivery (rate ratio [RR] = 0.6, 95% confidence interval (CI) = 0.4-0.9). An increased rate of preterm delivery occurred among women with Bacteroides bivius at concentrations of greater than 10(4) cfu/ml (RR = 2.0, 95% CI = 1.4-2.9) and Bacteroides fragilis (RR = 1.7, 95% CI = 1.0-2.8). Other microorganisms, including Gardernella vaginalis, the genital mycoplasmas, group B streptococci, Escherichia coli, and Peptostreptococcus species, were not associated with an increased rate of preterm delivery for women in preterm labor.

Relationship of bacteriologic characteristics to semen indices in men attending an infertility clinic.

Bacteria can be isolated from most seminal fluid samples, but the significance of these microorganisms is uncertain because most men lack symptoms associated with bacterial infection of the reproductive tract. We obtained semen samples from 37 men attending a Special Infertility Clinic and assessed the relationship between seminal fluid microorganisms and seminal fluid analysis including sperm motility, morphology, and concentration; the numbers of polymorphonuclear leukocytes and other white blood cells; and the hamster zona-free oocyte sperm penetration assay. Aerobic and/or anaerobic bacteria were recovered from 36 of the 37 samples. One hundred eighty-eight isolates (113 aerobes, 74 anaerobes, and one yeast) were recovered, with a mean of 5.2 isolates per semen specimen. The microorganisms recovered from the samples included: coagulase-negative staphylococci (89%), viridans streptococci (65%), diphtheroids (86%), Peptostreptococcus sp (62%), Bacteroides sp (27%), Gardnerella vaginalis (19%), Lactobacillus sp (16%), Actinomyces sp (16%), Enterococcus (11%), and Veillonella (11%). Other microorganisms including group B streptococcus, Hemophilus, Escherichia coli, Mobiluncus, and Clostridium were each recovered from fewer than 10% of the specimens. When the microbiology of seminal fluid specimens with or without polymorphonuclear leukocytes was compared, the presence of polymorphonuclear leukocytes in the semen was not associated with the isolation of staphylococci (33 versus 25%), viridans streptococci (33 versus 28%), Bacteroides sp (17 versus 37%), or Peptostreptococcus (31 versus 33%) (P greater than .05 for each comparison). The proportion of semen samples yielding bacterial isolates was similar after categorization by normal motility (more than 60%), pyospermia (six or more leukocytes per 100 sperm), sperm concentration, morphology, and a normal sperm penetration assay (11% or more).(ABSTRACT TRUNCATED AT 250 WORDS)