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Characterization of NAD-dependent 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and of NADP-dependent 7 beta-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 3 alpha- and 3 beta-hydroxysteroid dehydrogenases and a NADP-dependent 7 beta-hydroxysteroid dehydrogenase. All enzyme activities could be demonstrated in crude extracts and in membrane fractions. The 3 alpha- and 3 beta-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 3 alpha (3 beta)-hydroxysteroid dehydrogenase, a pH optimum of 8.5 (9.5) and a molecular weight of 95,000 (132,000) was estimated. 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were heat-sensitive (about 75% inactivation at 50 degrees C for 10 min). The 7 beta-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 7 beta-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 7 beta-hydroxysteroid dehydrogenase was heat-sensitive (72% inactivation at 50 degrees C for 10 min), but was detectable at 4 degrees C for at least 48 h.

3-Hydroxysteroid Dehydrogenases↗

Infection following challenge of the lactating and dry udder of dairy cows with Actinomyces pyogenes and Peptostreptococcus indolicus.

Challenge of 12 mammary glands of cows in mid-lactation with 10(7) colony forming units (cfu) of Peptostreptococcus indolicus on two occasions led to clinical mastitis in only four quarters. The bacteria were rarely recovered and disappeared from the secretion within 14 days. In challenges 7 days prior to drying off eight of 12 quarters became infected and at drying off all quarters challenged became infected. The infections established at drying off persisted well into the dry period. P. indolicus infection was also established in all of 12 dry glands challenged, but usually eliminated at calving or early in the next lactation. Isolation of P. indolicus was accompanied in about one-third of cases by changes in the appearance of the secretion. Intramammary challenge with Actinomyces (formally Corynebacterium) pyogenes led to clinical and subclinical infections in nine of 12 lactating glands and in all of six dry glands. Dry period infections with A. pyogenes were more severe and rarely eliminated even by antibiotic therapy. Infections during lactation were often eliminated either naturally or by antibiotic therapy. Intermittent recovery of A. pyogenes from the lactating mammary gland, without clinical signs of infection, was possible for up to 90 days after challenge. Combined infections with A. pyogenes and P. indolicus were clinically more severe with a higher frequency of systemic involvement. It was shown that in the non-lactating gland an acute mastitis, similar to 'summer mastitis' could be established either by simultaneous inoculation with A. pyogenes and P. indolicus or by subsequent inoculation of quarters excreting P. indolicus with A. pyogenes.

Actinomycosis↗

Polysaccharides from Peptostreptococcus anaerobius and structure of the species-specific antigen.

The cell-envelope antigens of Peptostreptococcus anaerobius were extracted from intact cells by autoclave or alkaline treatment. The purified species-specific antigen (G) was identified among several polysaccharides obtained from the extracts by successive treatments with ribonuclease and pronase followed by ion-exchange and gel-filtration chromatography. G was investigated by 13C- and 31P-n.m.r. spectroscopy, titrimetry, elemental analysis, and gas-liquid chromatography. Oxidation of G with NaIO4 followed by reduction with NaBH4 and mild acid hydrolysis yielded the Smith degradation product of G (GS). Treatment of G and GS with 48% HF gave the respective dephosphorylated products GF and GSF. The structures of GS, GF, and GSF were investigated by 13C-n.m.r. spectroscopy, methylation analysis, and gas-liquid chromatography-mass spectrometry. The principal constituents of G were 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), D-glyceric acid, and phosphate as a diester, in the ratio 2:1:1, and a minor amount of D-glucose (beta-D-Glcp). GS contained D-GlcNAc, D-glyceric acid, glycerol, and phosphate in a 1:1:1:1 ratio. GF and GSF contained D-GlcNAc and D-glyceric acid in the ratios 2:1 and 1:1, respectively. A structure for the principal repeating unit of polymeric G compatible with the analytical data consists of alpha-D-GlcpNAc-(1----3)-alpha-D-GlcpNAc-(1----2)-D-glyceric acid units linked through C-6'-C-6" phosphate diester bridges. This structure is novel for two reasons: (a) unsubstituted glyceric acid residues occur as aglycons in the repeating structure, and (b) phosphate diester bridges link nonanomeric glycose carbons in a non-nucleic acid polymer. The structural role of the minor amount of beta-D-Glcp in G remains unknown.

Antigens, Bacterial↗

Iron-sulfur cluster-containing L-serine dehydratase from Peptostreptococcus asaccharolyticus: correlation of the cluster type with enzymatic activity.

Investigations were performed with regard to the function of the iron-sulfur cluster of L-serine dehydratase from Peptostreptococcus asaccharolyticus, an enzyme which is novel in the class of deaminating hydro-lyases in that it lacks pyridoxal-5'-phosphate. Anaerobically purified L-serine dehydratase from P. asaccharolyticus revealed EPR spectra characteristic of a [3Fe-4S]+ cluster constituting 1% of the total enzyme concentration. Upon incubation of the enzyme under air the intensity of the [3Fe-4S]+ signal increased correlating with the loss of enzymatic activity. Addition of L-serine prevented this. Hence, active L-serine dehydratase probably contains a diamagnetic [4Fe-4S]2+ cluster which is converted by oxidation and loss of one iron ion to a paramagnetic [3Fe-4S]+ cluster, resulting in inactivation of the enzyme. In analogy to the mechanism elucidated for aconitase, it is proposed that L-serine is coordinated via its hydroxyl and carboxyl groups to the labile iron atom of the [4Fe-4S]2+ cluster.

Catalysis↗

Purification and characterization of NADP-dependent 7 beta-hydroxysteroid dehydrogenase from Peptostreptococcus productus strain b-52.

An NADP-dependent 7 beta-hydroxysteroid dehydrogenase was purified 11.5-fold over the activity in crude cell extracts prepared from Peptostreptococcus productus strain b-52, by using Sephadex G-200 and DEAE-cellulose column chromatography. 7 beta-Dehydrogenation was the sole transformation of bile acids catalyzed by the partially purified enzyme. The enzyme preparation (spec. act. 2.781 IU per mg protein) had an optimum pH of 9.8. Lineweaver-Burk plots showed a Michaelis constant (Km) value of 0.05 mM for 3 alpha, 7 beta-dihydroxy-5 beta-cholanoic acid whereas higher values were obtained with 3 alpha,7 beta-dihydroxy-5 beta-cholanoyl glycine (0.20 mM), and 3 alpha,7 beta-dihydroxy-5 beta-cholanoyl taurine (0.26 mM). NADP but not NAD could function as an electron acceptor, and had a Km value of 0.30 mM. A molecular weight of 64000 was determined by SDS-polyacrylamide gel electrophoresis. The addition of 0.4 mM of either bile acid to the growth medium suppressed not only cell growth, but also the enzyme yield.

Hydrogen-Ion Concentration↗

Sodium dependent acetate formation from CO2 in Peptostreptococcus products (strain Marburg).

Washed cells of Peptostreptococcus products (strain Marburg), which were incubated in the presence of CO/CO2/N2 (50%/17%/33%; 200 kPa) catalyzed the synthesis of acetate from carbon monoxide. The rate of acetate formation from CO was stimulated more than threefold by the addition of sodium (10 mM); potassium did not effect acetate synthesis. The degree of stimulation was dependent on the sodium concentration; the dependence followed simple Michaelis-Menten kinetics. The apparent Km for sodium was determined to be about 2 mmol/l. Sodium also stimulated acetate synthesis from H2 plus CO2. In the absence of added sodium the formation of formate as an intermediate in methyl group synthesis was stimulated. It is suggested that the sodium dependent reaction(s) is one (or more) of the reactions involved in methyl group synthesis from CO2.

Acetates↗

Comparative evaluation of the metabolic potentials of different strains of Peptostreptococcus productus: utilization and transformation of aromatic compounds.

Three strains of Peptostreptococcus productus were tested for growth at the expense of methoxylated aromatic compounds. Strain M8A-18 (human fecal isolate) was unable to utilize methoxylated aromatic compounds. While the type strain ATCC 27340 (human septicemia isolate) was capable of minimal growth with methoxylated aromatic compounds, ATCC 35244 (sewage sludge isolate) displayed significant growth on methoxylated aromatic compounds. Methoxylated phenols, benzoates, benzyl alcohol and phenylacrylates supported the growth of ATCC 35244 and were O-demethylated to their respective hydroxylated derivatives. During O-methyl- or CO-dependent growth, the double bond of the acrylate side chain of certain methoxylated and non-methoxylated phenylacrylates was reduced. Although other aromatic substituent groups (-COOH and -CH3) were transformed during CO-dependent growth, in short-term growth studies, the aromatic ring was not subject to reduction or degradation. Of the three strains tested, only strain M8A-18 failed to grow at the expense of carbon monoxide (CO).

Acrylates↗

Summer mastitis in heifers: studies on the seasonal occurrence of Actinomyces pyogenes, Peptostreptococcus indolicus and Bacteroidaceae in clinically healthy cattle in Denmark.

With the aim of investigating the seasonal occurrence of Actinomyces pyogenes, Peptostreptococcus indolicus, Bacteroides melaninogenicus ss. levii and Fusobacterium necrophorum, and thus the potential for development of summer mastitis, clinically healthy Danish Holstein-Friesian heifers due to calve in the autumn were sampled from the teat tip, the conjunctiva and the oral cavity at 2-6 week intervals from 1979 to 1981. The overall isolation rates of F. necrophorum, P. indolicus and B. melaninogenicus ss. levii, in order of significance, were significantly higher during the pasture period whereas no differences in isolation rates of A. pyogenes between housed and pastured animals were detected. F. necrophorum was recovered almost exclusively from the oral cavity, P. indolicus and A. pyogenes occurred most frequently in samples from the teat skin, whereas isolates of B. melaninogenicus ss. levii were evenly distributed between conjunctiva and teat tip samples. A distinct seasonal pattern of the isolation rates of summer mastitis pathogens was recorded, which corresponded closely to the seasonal activity of symbovine insects, in particular the headfly Hydrotaea irritans (Fallén). However, the high proportion of clinically healthy bacterial carriers as compared with the incidence of clinical disease strongly suggests that as yet unknown contributing or triggering factors, apart from the mere presence of the relevant bacterial species, are required for the establishment and development of clinical summer mastitis.

Actinomycosis↗

Micromonas (Peptostreptococcus) micros: unusual case of prosthetic joint infection associated with dental procedures.

Micromonas (Peptostreptococcus) micros is frequently associated with periodontal disease as well as respiratory, gastrointestinal and female genitourinary tract infections, but only rarely has been reported as a pathogenic agent of prosthetic joint infections. Here we describe a case of a 63-year-old woman with prosthetic joint infection of total hip arthroplasty caused by the anaerobic species Micromonas micros, associated with tooth extraction. Samples obtained intraoperatively and from the oral cavity were positive for the presence of M. micros by culture and by real-time PCR. This case report indicates that infections of prosthetic joints can be associated with dental procedures and that sensitive molecular techniques are necessary for their routine diagnostic.

Aged↗

Metastatic Peptostreptococcus intermedius endophthalmitis after a dental procedure.

A 46-year-old man developed symptoms of a chronic progressive uveitis in his right eye approximately one week after a dental procedure. The patient's intraocular inflammation was not diminished by massive treatment with topical and systemic corticosteroid therapy or intravenously administered adrenocorticotropic hormone. The inflammatory process progressed to an overt endophthalmitis during a period of three weeks and the eye eventually required evisceration. A pure culture of Peptostreptococcus intermedius was isolated from the eye. The most likely source of this organism was hematologic transport following a dental procedure.

Bacterial Infections↗

Splenic abscess caused by Peptostreptococcus species, diagnosed with the aid of abdominal computerized tomography and treated with percutaneous drainage and antibiotics: a case report.

We describe herein a 63-year-old patient with a splenic abscess due to Peptostreptococcus spp., diagnosed with the aid of abdominal computerised tomography and treated with ultra-sound guided percutaneous drainage and antibiotics. The bacteriological characteristics of splenic abscesses are discussed.

Abscess↗

Metabolism of pyrrolizidine alkaloids by Peptostreptococcus heliotrinreducens and a mixed culture derived from ovine ruminal fluid.

A mixed culture of ovine ruminal microbes metabolizes the macrocyclic pyrrolizidine alkaloids present in the plant Senecio jacobaea, including jacobine and seneciphylline. Previous attempts to identify metabolites of these alkaloids have not been successful. The objective of this study was to compare the metabolism of pyrrolizidine alkaloids by a mixed culture of ovine ruminal microbes to the metabolism of pyrrolizidine alkaloids by the known organism Peptostreptococcus heliotrinreducens. P. heliotrinreducens metabolizes the pyrrolizidine alkaloids heliotrine and lasiocarpine to 7alpha-hydroxy-1-methylene-8alpha-pyrrolizidine and 7alpha-angelyl-1-methylene-8alpha-pyrrolizidine, respectively. This organism does not metabolize the pyrrolizidine alkaloids jacobine or seneciphylline. A mixed culture of ovine ruminal microbes also metabolized heliotrine and lasiocarpine to identical methylene compounds. This mixed culture also metabolized jacobine and seneciphylline, with the production of very low levels of the corresponding 1-methylene compounds. Samples were analyzed by TLC and GC/MS.

Animals↗

Induction in rats of serum agglutinins to Eubacterium, Peptostreptococcus and Coprococcus species by the faecal flora from patients with Crohn's disease and healthy subjects.

Sera from patients with Crohn's disease more often than those from other subjects contain agglutinins against anaerobic coccoid rods, identified as Peptostreptococcus productus, Eubacterium contortum (two strains) and Coprococcus comes. The presence of antigens of the four coccoid rods in faeces of patients with Crohn's disease and healthy subjects was investigated by inoculation of rats with faecal suspensions. Antigens of the coccoid rods were detected in faeces from both patients with Crohn's disease and healthy subjects.

Agglutination Tests↗

Antibodies to Eubacterium and Peptostreptococcus species and the estimated probability of Crohn's disease.

Anaerobic coccoid rods belonging to species of Eubacterium and Peptostreptococcus agglutinate more frequently with sera from patients with Crohn's disease than with sera from patients suffering from other diseases and from healthy subjects. Results of agglutination test with four strains of coccoid anaerobes were used to estimate the probability that a patient suffers from Crohn's disease. The data on healthy subjects and patients with Crohn's disease were subjected to logistic discriminant analysis. With the methods and interpretation described, 52% of the patients with Crohn's disease were recognized as 'definite' or 'probable' Crohn's disease and 14% as 'suspected'. Only 1% of the healthy subjects were classified as 'suspected' and non as 'definite' or 'probable' Crohn's disease.

Adolescent↗

Adherence of Peptostreptococcus micros morphotypes to epithelial cells in vitro.

Peptostreptococcus micros, which is associated with oral and non-oral mixed anaerobic infections, occurs in three colony morphotypes, the smooth type, the rough type and the smooth variant of the rough type. These types differ in surface structures; the rough type expresses large fibrillar surface appendages, which are absent on the surface of both the smooth and the smooth variant of the rough type. To determine the role of these surface structures in adherence we characterized the adherence of the three morphotypes of P. micros to epithelial cells in vitro. Although all three types adhered well to epithelial cells, adhering numbers of the rough type were significantly lower than those of the smooth and the smooth variant of the rough type. Protease treatment increased the adherence of the rough type of the level of the two other types. The adherence of all three types was reduced more than 85% by treatment with 10 mM sodium periodate. Furthermore, the adherence was pH independent and could not be blocked by incubation with antisera to the bacteria. In addition, we determined the capacity to invade epithelial cells by P. micros. In an acridine orange assay such invasion could not be detected. Our results suggest that the adherence of P. micros to epithelial cells is mediated by periodate-sensitive extracellular polysaccharides and that the protruding fibril-like protein surface structures of the rough type have an obstructive effect on the adherence.

Adhesins, Bacterial↗

Tracer studies on the biosynthesis of amino acids from lactate by Peptostreptococcus elsdenii.

Peptostreptococcus elsdenii, a strict anaerobe from the rumen, was grown on a medium containing yeast extract and [1-(14)C]- or [2-(14)C]-lactate. Radioisotope from lactate was found in all cell fractions, but mainly in the protein. The label in the protein fraction was largely confined to a few amino acids: alanine, serine, aspartic acid, glutamic acid and diaminopimelic acid. The alanine, serine, aspartic acid and glutamic acid were separated, purified and degraded to establish the distribution of (14)C from lactate within the amino acid molecules. The labelling patterns in alanine and serine suggested their formation from lactate without cleavage of the carbon chain. The pattern in aspartic acid suggested formation by condensation of a C(3) unit derived directly from lactate with a C(1) unit, probably carbon dioxide. The distribution in glutamic acid was consistent with two possible pathways of formation: (a) by the reactions of the tricarboxylic acid cycle leading from oxaloacetate to 2-oxoglutarate, followed by transamination; (b) by a pathway involving the reaction sequence 2 acetyl-CoA-->crotonyl-CoA-->glutaconate-->glutamate.

Amino Acids↗

Enzymic studies on the biosynthesis of amino acids from lactate by peptostreptococcus elsdenii.

Cell-free extracts of Peptostreptococcus elsdenii, a strict anaerobe from the rumen, were examined for enzymes catalysing the steps in the biosynthesis from lactate of alanine, serine, aspartate and glutamate. Extracts contain the enzymes necessary for the formation of alanine from lactate via pyruvate. The presence of enzymes catalysing the interconversion of phosphoglycerate and phosphohydroxypyruvate, the transamination of the latter to phosphoserine and the cleavage of phosphoserine to serine and inorganic phosphate was demonstrated, suggesting that serine is formed via these intermediates. ;Malic' enzyme, malate dehydrogenase and glutamate-oxaloacetate transaminase are present in extracts and could account for aspartate formation. The extracts catalyse all of the steps of the tricarboxylic acid pathway leading from oxaloacetate plus acetate to glutamate. Together with substantive data from previous radioactive tracer studies the results provide strong evidence that these four amino acids are synthesized in this strict anaerobe by pathways closely similar to those operating in aerobic and facultatively aerobic organisms.

Alanine↗