Rainer Hammann

A Newly Discovered Sialidase from Gardnerella Vaginalis

A sialidase (neuraminidase, acylneuraminosyl hydrolase, EC 3.2.1.18) has been discovered and isolated from Gardnerella vaginalis (ex. Haemophilus vaginalis), a possibly pathogenic inhabitant of the female genital tract. Bacteria were grown in peptone-yeast-extract medium with 2.0 mM N-acetylmannosamine as enzyme inductor under CO2 atmosphere. Sialidase activity was found in the bacterial sediment and in the culture medium. The enzyme was liberated from the cells by ultrasonic treatment. Purification was performed by 60-80% ammonium sulfate precipitation and by column chromatography on Sepharose CL-6B and Sephadex G 200. The enzyme revealed a molecular weight in the range of Mr 75 000 and a pH optimum at 5.5. Among the different types of NeuAc-containing glycoconjugates, the enzyme exhibits its highest activities towards the globular glycoproteins alpha 1-acid glycoprotein and fetuin. Taking their cleavage rate as 100, it is around 55 for II3NeuAc-Lac, 45 for bovine submaxillary mucin, 35 for II6NeuAc-Lac and IV3, III6NeuAc2-LcOse4. The rates for III8,II3NeuAc2-Lac, gangliosides and colominic acid are below 20. Due to its specificity pattern, the enzyme may play a role in the pathogenic process of G. vaginalis infections.

Characterization of NAD-dependent 3α- and 3β-hydroxysteroid dehydrogenase and of NADP-dependent 7β-hydroxysteroid dehydrogenase from Peptostreptococcus productus

A human fecal isolate, characterized by morphological, physiological and biochemical data as a strain of Peptostreptococcus roductus, was shown to contain NAD-dependent 3α- and 3β-hydroxysteroid dehydrogenases and a NADP-dependent 7β-hydroxysteroid dehydrogenase. All enzyme activities could be demonstrated in crude extracts and in membrane fractions. The 3α- and 3β-hydroxysteroid dehydrogenases were synthesized constitutively. Specific enzymatic activities were significantly reduced when bacteria were grown in the presence of 3-keto bile acids, while other bile acids were ineffective. For the 3α(3β)-hydroxysteroid dehydrogenase, a pH optimum of 8.5 (9.5) and a molecular weight of 95 000 (132 000) was estimated, 3α- and 3β-hydroxysteroid dehydrogenases were heat-sensitive (about 75% inactivation at 50°C for 10 min). The 7β-hydroxysteroid dehydrogenase was already present in uninduced cells, but specific activity could be enhanced up to more than 2.5-fold when bacteria were grown in the presence of 7-keto bile acids. Disubstituted bile acids were more effective than trisubstituted ones, ursodeoxycholic acid was ineffective as an inducer. A pH optimum of 10.0 and a molecular weight of about 82 000 were shown for the 7β-hydroxysteroid dehydrogenase. The enzyme preparation reduced the 7-keto group of corresponding bile acids. Again the affinities of disubstituted bile acids for the enzyme were higher than those of the trisubstituted bile acids, but no significant differences between conjugated and free bile acids were observed. The 7β-hydroxysteroid dehydrogenase was heat-sensitive (72% inactivation at 50°C for 10 min), but was detectable at 4°C for at least 48 h.

NADP-dependent 3β-, 7α- and 7β-hydroxysteroid dehydrogenase activities from a lecithinase-lipase-negative Clostridium species 25.11.c

Abstract A lecithinase-lipase-negative Clostridium sp. 25.11.c., not fitting in any of the species of Clostridia described so far as judged by morphological, physiological, and biochemical data, was shown to contain NADP-dependent 3β-, 7α- and 7β-hydroxysteroid dehydrogenases. The three hydroxysteroid dehydrogenases could be demonstrated in the supernatant and in the membrane fraction after solubilization with Triton X-100, suggesting enzymes which were originally membrane bound. The 3β-hydroxysteroid dehydrogenase was synthesized constitutively, and the specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids and trisubstituted bile acids. A pH optimum of 7.5 and a molecular weight of approx. 104000 were estimated by molecular sieve chromatography. The enzyme reduced the 3-keto group of bile acids; an oxidation of a 3β-hydroxyl function could not be demonstrated. The lowest K m values were found for disubstituted bile acids, tribustituted and conjugated bile acids having higher K m values. 7α-Hydroxysteroid dehydrogenase, but not 7β-hydroxysteroid dehydrogenase, was already present in uninduced cells. The specific activities, however, were greatly enhanced when cells grown in the presence of chenodeoxycholic acid or 3α-hydroxy-7-keto-5β-cholanic acid. Ursodeoxycholic acid with its 7β-hydroxyl group was ineffective as an inducer. Molecular weights of approx. 82000 and 115000 were found for the 7α-hydroxysteroid dehydrogenase and the 7β-hydroxysteroid dehydrogenase, respectively. In contrast to the in vivo situation, the reaction could only be demonstrated in the reductive in vitro. Here, the pH optimum for the overall reaction was 8.5–8.7. 3β-, 7α- and 7β-hydroxysteroid dehydrogenase activities were readily demonstrated for at least 48 h when preparations were stored at 4°C, but were found to be heat-sensitive.

Falcivibrio grandis gen. nov. sp. nov., and Falcivibrio vaginalis gen. nov. sp. nov., a New Genus and Species to Accommodate Anaerobic Motile Curved Rods Formerly Described as “Vibrio mulieris” (Prévot 1940) Breed et at. 1948

Summary Strains of anaerobic, motile, curved rods, formerly designated “ Vibrio mulieris ”, wereisolated from human vaginal secretions. They were investigated for their biochemical, morphological and cytological behaviour and compared to similar strains from other authors. The strains studied were divided into two species on the basis of their physiologicalactivities and morphology. The first species comprises saccharolytic strains with a crescent cell shape and a GC base ratio of 50-53, the second species asaccharolytic strains with a less crescent, but often coryneform cell shape and a GC base ratio of 53-54. The Gram reaction of the organisms is variable. Electron microscopy of the cell wallcould not elucidate the Gram type clearly. The saccharolytic strains are described as Falcivibrio grandis gen. nov. sp. nov., theasaccharolytic strains as Falcivibrio vaginalis gen. nov. sp. nov. The new genus cannot be assigned to any of the recognized families.

Quantitative studies on the vaginal flora of asymptomatic women and patients with vaginitis and vaginosis.

Vaginal washings of 22 patients with vaginitis, 11 with vaginosis, and 12 healthy subjects were investigated quantitatively and qualitatively for aerobic and anaerobic bacteria and yeasts. Gardnerella vaginalis was recovered from 9 of the vaginitis patients, 7 of the vaginosis patients, and 4 of the asymptomatic subjects. Obligate anaerobes were found in 11 of the vaginitis patients, 4 of the vaginosis patients, and none of the control subjects. Bacteroides bivius was the anaerobe most frequently isolated from symptomatic subjects. Anaerobic vibrios were recovered twice from symptomatic subjects. The counts for Gardnerella vaginalis and anaerobes when present were generally very high. The most frequent aerobes were beta-hemolytic streptococci (group B) and staphylococci.

Occurrence of bacteroidaceae in vaginal secretions of healthy pregnant women and patients with colpitis

SummaryThe frequency of gram-negative non-sporing anaerobes with possible clinical significance was studied in healthy pregnant women and in patients with non-Candida vaginitis. Only 4% of the healthy pregnant women studied yielded anaerobes of the familyBacteroidaceae. However, the incidence ofBacteroidaceae in 58 patients with colpitis was 53.5%. The predominant anaerobic species wasBacteroides oralis.ZusammenfassungBei gesunden Schwangeren und bei Patientinnen mit nicht durchCandida bedingter Kolpitis wurde die Häufigkeit von gramnegativen sporenlosen Anaerobiern mit möglicher klinischer Bedeutung untersucht. Nur bei 4% der untersuchten gesunden Schwangeren waren Anaerobier der FamilieBacteroidaceae nachweisbar. Dagegen betrug bei 58 Kolpitis-Patientinnen dieBacteroidaceae-Inzidenz 53,5%. Die vorherrschende Anaerobierart warBacteroides oralis.

Isolation, characterization and ecological distribution of recently recognized propionic acid-negative Bacteroides species from human sources

140 strains of anaerobic, Gram-negative, propionate-negative, non-pigmented rods (Bacteroides oralis group) were isolated from normal flora and from clinical specimens. They were characterized by physiological tests, gas chromatography of the acid end products, and determination of the G + C content of their DNA. In addition, their distribution in the various biotopes of the human body (oropharyngeal tract, genitourinary tract, and gut) and in clinical specimens was investigated. The strains could be assigned to the recently described species Bacteroides denticola, B. buccae, B. buccalis, and B. oris, or to B. bivius, B. disiens and B. oralis. In the investigated group, especially B. bivius exhibits an antibiotic resistance--particularly against the beta-lactam antibiotics--comparable to that of B. fragilis.

Thin Layer Chromatography for Rapid Detection of Carbohydrate Utilization by Bacteroides Strains

19 weakly saccharolytic Bacteroides strains of different species were tested by thin-layer chromatography (TLC) for their fermentative abilities for fructose, sucrose, lactose, galactose, and raffinose. Conventional fermentation tests were run parallel. In general, a good agreement between both methods was recorded. Two strains, however, showed a degradation in the TLC test without an acidification. With some strains, sucrose as a substrate yielded a fructose spot, lactose a galactose spot, and raffinose a melibiose spot, indicating an incomplete degradation of these carbohydates.

CO2-abhängige Mutante von Salmonella typhi als Ursache typhöser Erkrankungen bei zwei Patienten

Abstract A CO 2 -dependant mutant of Salmonella typhi was isolated from anaerobic but not from aerobic blood-cultures of two patients with typhoid fever. Both patients diseased after return from their home country Turkey. The strains developed in a CO 2 -enriched atmosphere and under anaerobic but not aerobic culture conditions. This property remained stable during 20 subcultures. The fermentative and serological pattern was identical with S. typhi T 2 . Both strains were of lysovar E 1a .

Neuraminidases of Bacteroidaceae

Abstract Neuraminidases can be detected in members of the anaerobic gram-negative non-sporing rods (Bacteroidaceae), especially in the genus Bacteroides. B. fragilis, the most virulent species, has the highest neuraminidase activity, while the other intestinal species exhibit markedly lower activities or the enzyme is completely absent. Members of the Bacteroides oralis group, so far investigated, degrade only substrates of lower molecular weight. Zusammenfassung Bei den anaeroben gram-negativen Stabchenbakterien (Familie Bacteroidaceae) kommen Neuraminidasen hauptsachlich in der Gattung Bacteroides vor. B. fragilis, die Art mit der starksten Pathogenitat, hat die starkste Neuraminidaseaktivitat, wahrend die anderen intestinalen Bacteroides-Arten keine oder nur niedrige Aktivitatsraten aufweisen. Die bisher untersuchten Bacteroides-Arten aus der B. oralis-Gmppe bauen im wesentlichen nur Substrate mit niedrigem Molekulargewicht ab.