Relationship between hydrogen peroxide-producing strains of lactobacilli and vaginosis-associated bacterial species in pregnant women.

This study was conducted to determine the relationship between lactobacilli and bacterial species associated with bacterial vaginosis in pregnancy and the prevalence of H2O2-producing and non-producing strains of lactobacilli in pregnant women whose vaginal flora had already been analysed. Information was available for 174 pregnant women whose vaginal flora had been evaluated previously by examining gram-stained vaginal smears: 50 had grade III flora (bacterial vaginosis). 50 grade II flora, 41 flora graded as abnormal which then reverted to grade I (revertants) and 33 normal flora (controls). Lactobacilli were isolated from 19 of 50 women whose vaginal flora was grossly abnormal culturally and categorised as grade III by Gram staining. In 6 of these 50 women lactobacilli were isolated in large numbers, i.e. 10(5)-10(6) cfu/ml. H2O2-producing strains of lactobacilli were isolated from 11 of 12 women with grade III flora who were randomly selected from this group. Thus, in those 11 women it appears that H2O2-producing lactobacilli had not protected them from developing bacterial vaginosis. Bacterial species associated with vaginosis were isolated in high numbers from a large proportion of women in the revertant and grade II groups in association with high counts of lactobacilli. Thus, in some women it is possible that a change to an abnormal flora could occur before the complete disappearance of lactobacilli. It is concluded that bacterial vaginosis may develop in some women despite the presence of H2O2-producing strains of lactobacilli and that other factors, as yet unidentified, might be conducive to the appearance of abnormal bacterial flora with progression to vaginosis.

A placebo-controlled, double-blind prospective study in healthy female volunteers of dextrin sulphate gel: a novel potential intravaginal virucide.

A double-blind, placebo-controlled study was designed to evaluate the safety and tolerability of intravaginal dextrin sulphate (D2S) gel to assess its preliminary suitability as a potential vaginal virucide. Tolerability was assessed by questionnaire and patient interview. Colposcopy with vaginal biopsy was performed to assess the macroscopic and microscopic evidence of inflammation. The potential impact of the gel on normal vaginal flora was examined by quantitative lactobacilli culture with assessment of the ratio of peroxide to nonperoxide-producing organisms. Colposcopy revealed mild erythema in five of 24 subjects receiving active gel and in none of the 12 placebo recipients, but histology in all subjects revealed no evidence of inflammation. No impact on vaginal lactobacilli was found. We conclude that D2S gel is safe and well tolerated intravaginally at the dosing schedule used in this study.

Comparison of quantitative and qualitative methods of detecting hydrogen peroxide produced by human vaginal strains of lactobacilli.

A quantitative method was developed for the measurement of micromolar quantities of H2O2 produced in Rogosa broth and peptonized milk broth by vaginal strains of lactobacilli isolated from women. The production of substantial amounts reproducibly was dependent on the growth of the organisms in acid media (pH less than or equal to 6.0) under anaerobic or micro-aerophilic conditions with continuous agitation. The addition to the media of the enzyme inhibitor, 3-amino-1,2,4-triazole, with or without catalase sometimes induced the production of H2O2 especially in non-agitated cultures. However, other agents such as concanavalin and o-dianisidine had no enhancing effect, and catalase or peroxidase alone completely inhibited H2O2 production. The H2O2 produced in the acid media was stable for more than a month at 5 degrees C but not in media at pH greater than or equal to 7.0. Of five strains of lactobacilli tested by the quantitative method and by a chromogenic qualitative method (Rogosa-catalase or -per-oxidase agar), three consistently produced H2O2 measurable by the former method, but none did so after growth of the organisms on Rogosa-catalase/peroxidase agar which suggested that the qualitative method was unreliable. The fact that H2O2 was produced in substantial quantities by some strains and not at all by others enabled H2O2-producers and non-producers to be distinguished easily.

The effect of a toxin from Bacteroides ureolyticus on the mucosal epithelium of human and bovine oviducts.

Bacteria-free filtrates of nine strains of Bacteroides ureolyticus, most of which had been isolated from the urethra of men with non-gonococcal urethritis, damaged the mucosal epithelium of human fallopian tube and bovine oviduct organ cultures. The damage, pronounced after three days, was manifested by loss of ciliary activity. Histological observations, supported by scanning electron microscopy, showed that this loss was due to disruption of the epithelia with sloughing of cells. It is likely that the inhibitory activity of the filtrates was due to endotoxin since lipopolysaccharides extracted from the bacteria had a similar deleterious effect on oviduct mucosal epithelia. It is speculated that B. ureolyticus has the potential for causing damage to the urethral mucosa by the same mechanism.

The enigma of non-gonococcal urethritis: role for Bacteroides ureolyticus.

Although up to about half the cases of acute non-gonococcal urethritis (NGU) are caused by Chlamydia trachomatis organisms (chlamydiae) and a smaller, ill-defined, proportion probably by Ureaplasma urealyticum organisms (ureaplasmas), the aetiology of all cases is not understood. Clarification of the role of the anaerobe, Bacteroides ureolyticus, was sought in the current study. Seventy five chlamydia negative patients with NGU were treated on a double blind placebo controlled basis with metronidazole. After seven days more of the 35 patients given this drug tended to improve clinically than the 40 given the placebo, but the difference was not significant. Of 23 chlamydia negative but anaerobe positive men, however, 78% (7/9) receiving metronidazole responded clinically, but only 7% (1/14) receiving placebo responded (p less than 0.001). Furthermore, whereas 78% of the anaerobe positive men given metronidazole recovered, only 23% (6/26) of the anaerobe negative men did so (p less than 0.02). No further evidence for the role of ureaplasmas in the aetiology of NGU was obtained, but the data suggest that B ureolyticus organisms, and perhaps other anaerobes, have an important role in a small proportion of cases and that the beneficial effects of metronidazole given on an empirical basis will be confined to anaerobe positive urethritis.

A numerical taxonomic study of anaerobic gram-negative bacilli classified as Bacteroides ureolyticus isolated from patients with non-gonococcal urethritis.

A numerical taxonomic study of 64 strains of anaerobic Gram-negative bacilli isolated from men with non-gonococcal urethritis, two unclassified laboratory strains of 'corroding bacilli', and 12 other strains of anaerobic Gram-negative bacilli, including nine received as anaerobic curved rods and three as 'Bacteroides corrodens' (B. ureolyticus), isolated from women with bacterial vaginosis, was undertaken. Seventeen reference anaerobic strains belonging to the genera Bacteroides, Fusobacterium, Mobiluncus, Mitsuokella and Wolinella were included. Morphological, biochemical and physiological characteristics were examined in 103 tests. The resemblance between the 95 strains was calculated using the SSM, SJ and DP coefficients for cluster analyses based on the UPGMA method. All three approaches gave similar groupings, and the estimated average probability of test error was 2.46%. The strains fell into 10 phenons. The unclassified strains from men and three from women with lower genital-tract infections, and the laboratory strains of 'corroding bacilli' clustered in one phenon with the reference strains of B. ureolyticus, indicating that they correspond to B. ureolyticus. The other unclassified strains of anaerobic curved rods clustered as a distinct phenon. They correspond to species of the newly described genus Mobiluncus. The taxonomic data and the compilation of diagnostic tables serve as a useful guide for the laboratory identification of clinical isolates regarded as B. ureolyticus.

Characteristics of a gram-negative anaerobe isolated from men with non-gonococcal urethritis.

A small, fastidious gram-negative anaerobe was isolated from men with non-gonococcal urethritis (NGU). The isolates are described as NGU-associated anaerobes because they were extremely rare in men with urethritis other than NGU, and in asymptomatic men. They showed twitching motility, had many polar pili and appeared to be a homogenous group culturally, morphologically and biochemically. None of the strains fermented or utilised carbohydrates or organic acids as sole sources of carbon for energy and growth. However, growth of all strains was stimulated by formate and fumarate in liquid and solid media, especially in the former where growth seemed dependent on these growth factors. Unlike most anaerobes they produced cytochrome enzyme(s) that might be involved in oxidation-reduction reactions in the presence of oxygen as some of the strains were capable of growing in 5% oxygen. However, growth and energy generally resulted from anaerobic phosphorylation. Strains of this anaerobe seemed to require a low redox-potential (Eh) for survival during transportation but this was not essential for growth. Comparative studies with the other asaccharolytic anaerobes showed some similarity between the NGU-associated anaerobe, Bacteroides ureolyticus and Wolinella succinogenes. Like these, some NGU-associated strains pitted agar media and all produced urease. However, unlike these anaerobes, strains of the NGU-associated anaerobe produced enzymes for the hydrolysis of arginine, and the decarboxylation of lysine and ornithine. They also produced oxidase and some strains haemolysed sheep red cells. However, lactic acid was not an end-product of the metabolism of glucose by any of the strains. The NGU-associated anaerobes are strikingly different from anaerobic vibrios, B. praeacutus and B. asaccharolyticus.

Differential characteristics of a small gram-negative anaerobe associated with non-gonococcal urethritis which morphologically resembles Bacteroides ureolyticus.

Strains of a small, Gram-negative anaerobe described as 'NGU-associated' because of its apparent association with non-gonococcal urethritis (NGU), grew synergistically with anaerobic cocci on primary isolation. Subsequently, it was found that the addition of formate-fumarate to the media enhanced their growth. The organisms were asaccharolytic and, unlike most anaerobes, produced oxidase (cytochrome c) and tolerated 5% oxygen when grown in an atmosphere of 2% hydrogen, 7.5% carbon dioxide and 85.5% nitrogen. The strains examined appeared homogeneous culturally, morphologically and biochemically, and had some characteristics in common with Bacteroides ureolyticus (NCTC 10948) and Wolinella succinogenes (ATCC 29543). However, the latter was flagellated and had a DNA G + C content (46-49 mol%) that was different from B. ureolyticus (28 mol%) and the NGU-associated strains (28-30 mol%). These strains and B. ureolyticus shared many properties but electron microscopy of the NGU-associated strains revealed polar pili which were not seen on the strain of B. ureolyticus examined. Other tests which differentiated B. ureolyticus from the NGU-associated strains, being positive only for the latter, were lysine and ornithine decarboxylation, beta-haemolysis and growth in 5% oxygen: those that were negative for the NGU-associated strains were pitting of agar on primary culture and the production of acetate and lactate as metabolic end-products. Despite these differences, the NGU-associated strains may be biotypes or variants of B. ureolyticus, a species which we now consider needs further study and classification.

Development and evaluation of a selective medium for an anaerobe associated with non-gonococcal urethritis.

The purpose of the study was to develop a selective medium for the isolation of an anaerobe that is associated with non-gonococcal urethritis. This was achieved by determining the sensitivity of 64 strains of the NGU-associated anaerobe to 20 antibiotics and three dyes by the disc-diffusion method. Then the MIC of eight of these antimicrobial agents for ten of the strains was determined by a tube dilution method. The results led to the formulation of a selective agar medium based on the incorporation of polymyxin (0.15 IU/ml), trimethoprim (10 micrograms/ml), vancomycin (10 micrograms/ml) and methyl green (32 micrograms/ml). This medium supported growth of the NGU-associated anaerobe but did not support growth of the other anaerobes tested. The selective nature of the medium made possible the recovery of the NGU-associated anaerobe from 13 of the 25 specimens from men with NGU, compared with isolation from only 8 of these men when using a non-selective medium.

Anaerobes in men with urethritis.

Sixty-four men with non-gonococcal urethritis (NGU), seven with gonococcal urethritis (GU), and 30 who had no symptoms or signs of urethritis were studied. Chlamydia trachomatis was isolated from urethral specimens taken from 22% of the men with NGU, and 18% with GU, but not from those who did not have urethritis even though 20 (67%) of them had a history of NGU, GU, or both. The chlamydial isolation rate for men having NGU for the first time was 30%. Ureaplasma urealyticum was isolated from 42% of the men with NGU, 43% of men with GU, and 27% of those without urethritis.In addition to aerobes anaerobes were isolated frequently from men whether or not they had urethritis, the most common being anaerobic Corynebacteria, peptococci, and micro-organisms of the Bacteroides-Fusobacteria group. There was no appreciable difference in the overall isolation of anaerobes from men with NGU (89%) or from those without disease (80%). The rate of isolation of a Gram-negative anaerobic bacillus from men with NGU (50%) was, however, strikingly different to that from men with GU (14%) or from those without disease (13%). Furthermore, this bacillus was recovered from 28 (56%) of 50 men with NGU who were considered chlamydia-negative and from 19 (61%) of 31 men who were both chlamydia-negative and ureaplasma-negative. The effect of antibiotic treatment was evaluated in a few patients only, so that although those from whom the Gram-negative bacillus was eliminated by tetracycline recovered clinically there is a need for a prospective therapeutic trial to evaluate further the importance of the bacillus.