Effects of multispecies probiotic supplementation on intestinal microbiota in irritable bowel syndrome.

BACKGROUND: A multispecies probiotic has shown beneficial effects in irritable bowel syndrome. In addition, certain other probiotics have demonstrated advantageous effects, but the mechanisms behind this are poorly understood. AIM: To investigate the mode of action of a multispecies probiotic consisting of Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99 by monitoring its effects on intestinal microbiota and markers of microbial activity. METHODS: A total of 55 irritable bowel syndrome patients participated in this placebo-controlled double-blind trial. Subjects received either multispecies probiotic or placebo supplementation daily during a 6-month period. The composition of intestinal microbiota was analysed with real-time polymerase chain reaction, short-chain fatty acids with gas chromatography and enzymes with spectrophotometer. RESULTS: Each supplemented probiotic strain was detected in faecal samples. Intestinal microbiota remained stable during the trial, except for Bifidobacterium spp., which increased in the placebo group and decreased in the probiotic group (P = 0.028). No changes in short-chain fatty acids occurred. A decrease in ss-glucuronidase activity was detected in 67% of the subjects in the probiotic group vs. 38% in the placebo group (P = 0.06). CONCLUSIONS: Factors other than the microbial groups and metabolites studied herein seem responsible for the alleviation of irritable bowel syndrome symptoms by the multispecies probiotic.

High level heterologous protein production in Lactococcus and Lactobacillus using a new secretion system based on the Lactobacillus brevis S-layer signals.

A secretion cassette, based on the expression and secretion signals of a S-layer protein (SlpA) from Lactobacillus brevis, was constructed. E. coli beta-lactamase (Bla) was used as the reporter protein to determine the functionality of the S-layer signals for heterologous expression and secretion in Lactococcus lactis, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus gasseri and Lactobacillus casei using a low-copy-number plasmid derived from pGK12. In all hosts tested, the bla gene was expressed under the slpA signals and all Bla activity was secreted to the culture medium. The Lb. brevis S-layer promoters were very efficiently recognized in L. lactis, Lb. brevis and Lb. plantarum, whereas in Lb. gasseri the slpA promoter region appeared to be recognized at a lower level and in Lb. casei the level of transcripts was below the detection limit. The production of Bla was mainly restricted to the exponential phase of growth. The highest yield of Bla was obtained with L. lactis and Lb. brevis. Without pH control, substantial degradation of Bla occurred during prolonged cultivations with all lactic acid bacteria (LAB) tested. When growing L. lactis and Lb. brevis under pH control, the Bla activity could be stabilized also at the stationary phase. L. lactis produced up to 80 mg/l of Bla which to our knowledge represents the highest amount of a heterologous protein secreted by LAB so far. The short production phase implied a very high rate of secretion with a calculated value of 5 x 10(5) Bla molecules/cell per h. Such a high rate was also observed with Lb. plantarum, whereas in Lb. brevis the competition between the wild type slpA gene and the secretion construct probably lowered the rate of Bla production. The results obtained indicate wide applicability of the Lb. brevis slpA signals for efficient protein production and secretion in LAB.

Sequence analysis, distribution and expression of an aminopeptidase N-encoding gene from Lactobacillus helveticus CNRZ32 [gene 155 (1995) 89-93].

Lactobacillus (Lb.) helveticus CNRZ32 possesses a 97-kDa metalloenzyme with aminopeptidase activity (PepN; EC 3.4.11.2). A 3.8-kb fragment encoding PepN was cloned into pIL253 and designated pSUW34. Transformation of Lactococcus (Lc.) lactis LM0230 with pSUW34 resulted in > 180-fold increase in general aminopeptidase (AP) activity using L-lysine-p-nitroanilide. Southern hybridization was conducted to determine the distribution of homology to the CNRZ32 pepN gene among lactic-acid bacteria (LAB). Hybridization was observed with strains of lactobacilli, pediococci, leuconostoc, streptococci and lactococci. The pepN gene was sequenced and found to encode a protein containing 844 amino acid (aa) residues. A comparison of Lb. helveticus CNRZ32 pepN to Lb. delbrueckii ssp. lactis DSM7290 pepN indicated 69.5% nucleotide (nt) identity and 71.8% aa identity, while comparison to pepN from Lc. lactis ssp. cremoris MG1363 indicated 61.1% nt identity and 49.2% aa identity. Alignment of peptidase aa sequences of LAB, Escherichia coli, yeast and mammalian origin display homology in the zinc-binding domain, as well as a conserved region upstream from the putative active site.

Sequence analysis, distribution and expression of an aminopeptidase N-encoding gene from Lactobacillus helveticus CNRZ32.

Lactobacillus (Lb.) helveticus CNRZ32 possesses a 97-kDa metalloenzyme with aminopeptidase activity (PepN; EC 3.4.11.2). A 3.8-kb fragment encoding PepN was cloned into pIL253 and designated pSUW34. Transformation of lactococcus (Lc.) lactis LM0230 with pSUW34 resulted in > 180-fold increase in general aminopeptidase (AP) activity using L-lysine-p-nitroanilide. Southern hybridization was conducted to determine the distribution of homology to the CNRZ32 pepN gene among lactic-acid bacteria (LAB). Hybridization was observed with strains of lactobacilli, pediococci, leuconostoc, streptococci and lactococci. The pepN gene was sequenced and found to encode a protein containing 844 amino acid (aa) residues. A comparison of Lb. helveticus CNRZ32 pepN to Lb. delbrueckii ssp. lactis DSM7290 pepN indicated 69.5% nucleotide (nt) identity and 71.8% aa identity, while comparison to pepN from Lc. lactis ssp. cremoris MG1363 indicated 61.1% nt identity and 49.2% aa identity. Alignment of peptidase aa sequences of LAB, Escherichia coli, yeast and mammalian origin display homology in the zinc-binding domain, as well as a conserved region upstream from the putative active site.

Sandwich hybridization as a convenient method for the detection of nucleic acids in crude samples.

A method based on three-DNA-component, sandwich hybridization has been designed for the detection and quantitation of nucleic acids in crude samples using adenovirus DNA as a model. Two non-overlapping restriction fragments of adenovirus type 2 (Ad2) DNA were cloned into two vectors, the pBR322 plasmid and M13 phage. The recombinant plasmid DNA was immobilized onto nitrocellulose filters and the single-stranded recombinant phage DNA was labeled with 125I and used as a probe. When these two reagents were incubated under annealing conditions no radioactivity became filter-bound; only if denatured adenovirus DNA was added as the third reagent, it mediated the attachment of the radioactive probe to the filters. Hybridization efficiency was shown to be dependent on both the filter and probe DNA concentrations and on the hybridization conditions. When standardized, the assay is quantitative, and under the conditions used 0.2 ng of adenovirus DNA (8 X 10(-6) pmol) could be detected by an overnight incubation. The test is suitable for crude samples, e.g., solubilized cell extracts, without any purification steps. Less than 100 cells infected with Ad2 can be detected, implying that the assay could be applicable to virus diagnostics.

The secretory S complex in Bacillus subtilis is identified as pyruvate dehydrogenase.

We have cloned the operon for the Bacillus subtilis S complex, which has been suggested to be a component of the protein secretion machinery. The S-complex operon was found to encode 4 proteins, which were identified as subunits of pyruvate dehydrogenase (PDH). The Staphylococcus aureus membrane-bound ribosome protein (MBRP) complex has been considered to be a counterpart of the B. subtilis S complex. Here, we sequenced a fragment of the MBRP operon encoding the C-terminal part of E1 beta, the entire E2 and the N-terminal part of the E3 subunit of PDH, thus conclusively confirming the PDH identity of the MBRP complex as well. It appeared unlikely that PDH could be a primary component in protein secretion, thus disproving the previous hypothesis of the role of the S complex. However, attachment of the S complex (PDH) to the membrane and ribosomes may produce a biologically significant interaction.

Characterization and expression of the pepN gene encoding a general aminopeptidase from Lactobacillus helveticus.

An aminopeptidase N (pepN) gene was detected by DNA hybridization from an industrially important Lactobacillus helveticus strain using part of the L. helveticus CNRZ32 pepN gene as the probe. One of five hybridization positive clones was characterized in more detail. A subcloned 3.7 kb fragment, positive in hybridization and encoding aminopeptidase activity, was sequenced and analyzed. Only one open reading frame (ORF) of 2532 base pairs with a coding capacity for a 95.9 kDa protein could be found. The deduced amino acid sequence of the 95.9 kDa protein showed homology to PepN proteins from other lactic acid bacteria and carried the conserved catalytic and zinc binding sites of the neutral zinc metallo-peptidase family confirming the identity of the pepN gene. A 2.75 kb transcript and two transcription start sites were identified with mRNA analyses. Expression of pepN in L. helveticus, studied as the function of growth, revealed a high level of pepN transcripts throughout the growth, in contrast to the steady state levels of other peptidase mRNAs from L. helveticus analyzed in our laboratory.

Production and secretion of pertussis toxin subunits in Bacillus subtilis.

Pertussis toxin (PT) is a major component of today's acellular whooping cough vaccines. The use of acellular vaccines is predicted to increase sharply in the near future. There is therefore a need to produce PT in a way that makes its purification as easy as possible. Our approach was to express all five PT subunits individually in Bacillus subtilis. We have used vectors containing the promoter and signal sequences of the alpha-amylase gene of Bacillus amyloliquefaciens followed by an insert encoding the appropriate PT-subunit. All PT-subunits were secreted and found in the culture supernatant. The level of expression varied considerably: S1 and S5 were produced in large quantities whereas much smaller amounts of S2, S3 and S4 were found. The subunits were also present in the membrane fraction of the respective strains.

Microbial diagnosis by nucleic acid sandwich hybridization.

Sandwich hybridization is a three-component nucleic acid hybridization method suitable for the identification of microbes. In this method, one specific DNA fragment on solid support acts as catching reagent, and the second reagent is a labeled probe. The labeling of the support is mediated by a specimen nucleic acid homologous to both reagents. Because the specimen is kept in solution, relatively crude specimens not requiring elaborate pretreatments can be tested without background problems. The utility of the method in microbial diagnosis (adenovirus, cytomegalovirus, and Chlamydia trachomatis) has been demonstrated. Increased sensitivity and nonradioactive detection methods will no doubt further extend the applicability of the sandwich hybridization method.

Cryocauterization in the treatment of subglottic hemangioma in infants.

Cryocauterization seems to be an effective method of treating the subglottic hemangioma of infants. A series of five patients is presented. Cryotherapy, at temperatures of --40 degrees C or --70 degrees C for 60 or 30 seconds respectively, was given at intervals of 2 weeks, 3-6 sessions being required. during this time the child usually had to remain tracheostomized. No therapeutic or late complications were recorded. The patients have been followed up from 8 to 3 1/2 years, and none of them have shown disturbances of laryngeal development or predisposition to stricture.

Mucosal histochemistry in secretory otitis.

Mucosal biopsies were taken from 20 ears with secretory otitis media (glue ear) and histochemical stainings were made for comparison with data obtained from biochemical analysis of the fluids. Acid phosphatase, lactate dehydrogenase (LD), and malate dehydrogenase (MD), the activity of which in the ear fluids was 20 to 30 times higher than in serum, were found to appear as strong precipitates in the middle ear epithelium, particularly in the top layer. Alkaline phosphatase activity was only exceptionally seen in the epithelium but appeared in the capillaries and histiocytes. Nonspecific esterase appeared irregularly in the epithelium and regularly in histiocytes. The latter two had lower activities in ear fluids than in serum. Epithelial secretory cells and subepithelial glands and cysts showed strong alcian blue (AB)-positive staining. Positive material appeared also in the cytoplasm of the epithelial cells and in the intercellular substance. Distinct PAS-positive staining appeared in the columnar epithelium and particularly in the free mucus on top of the epithelium but was less promounced in the glandular structures and absent from the cysts.

Identification of a gene cluster for the mevalonate pathway in Lactobacillus helveticus.

Three Lactobacillus helveticus 53/7 genes essential for the biosynthesis of isopentenyl diphosphate and the gene coding for a putative carotenoid biosynthesis protein were for the first time identified from lactic acid bacteria. The deduced amino acid sequences of the mevalonate pathway gene products share significant identity with corresponding proteins of a few gram-positive cocci and Streptomyces species.

The role of the binaural test in filtered speech audiometry.

The value of the binaural resynthesis test administered according to Matzker's principle can be estimated only when a comparable monaural test can be used as a control. The most common positive finding in the filtered speech test is asymmetrical discrimination. Both parts of the message delivered to either ear must therefore have the same intelligibility in normal material. Binaural intelligibility is highly resistant to degenerative changes in the auditory system, for example. Positive findings in the binaural test are encountered frequently in brain stem lesions with vascular or traumatic aetiology.

Undistorted and filtered speech audiometry in children with normal hearing.

Undistorted and filtered speech tests were carried out in 140 test subjects with normal hearing acuity ranging in age from 4 to 19 years. Routine adult word lists were used in all tests. The intelligibility of undistorted speech rose markedly in the age groups from 4 to 9 years and correspondingly in the filtered speech test up to 11 years, taking the group 16-19 years as basis of reference. According to these results a speech discrimination test can be made in childhood with the same methods as in adults, if the norms are taken into account. In the filtered speech test the binaural test result and the monaural discrimination ability of the left ear were significantly better than the monaural test results for the right ear up to the age of 12-13 years.

Surgical treatment of Meniere's disease.

Results are reported of the effect ov various surgical methods on vertigo in Meniere's disease. Minor preocedures like sacculotomy or application of NaCl crystals in the round window may effect a cure, but often results in deaf ears. Vestibular nerve section seems to offer the best of vertigo, while generally still preserving residual hearing.

Otosclerosis surgery.

Results of otosclerosis surgery using the stapes footplate removal with various types of reconstruction are reported in 456 ears (360 patients). Primary success was obtained in around 95% while 83% retained their improvement over the years. The posterior crus on fascia has become the preferred method because of its stability and relative lack of inner ear complications. Inner ear damage is mostly due to perilymph fistulae, which should be promptly recognized and repaired, since revision after the ear has become deaf does not restore hearing. Some late inner ear losses may be due to reactions to foreign material introduced with the graft. Bilateral operations should not be done if the first operation was difficult or caused vertigo for several days.

Recruitment in conduction deafness. Observations using a midline loudness balance test.

A midline loudness balance test was made in 10 normal ears with blocked ear canals and in 104 conductively deaf ears with loss not exceeding 45 dB. The test was made by giving the tones simultaneously in the ears at 10, 60 and 80 dB sensation levels for 500 and 2 000 Hz. In the normal group the midline balance method indicated no reduction from the threshold differences at any of the three levels whereas significant reductions occurred in all groups of conduction deafness. The occurrence of true partial recruitment in conduction deafness demonstrated by ABLB (Fowler's) test was attributed to altered middle ear mechanics, loud tones stimulating the inner ear relatively more effectively than tones near the threshold level. The midline balance method is more closely dependent upon bone conducted sounds and on the altered relative movements of the labyrinthine windows.

Immunological defects in children with chronic otitis media.

A total of 59 children with chronic otitis media were examined for possible immunological defects by determination of serum gammaglobulin and immunoglobulins IgG, IgA and IgM, and by a tuberculin test, and additionally by noting any lymphopenia. Some divergence from normal values was found in 38 patients (64%). 14 children (24%) showed pathologically low immunoglobulin levels and 7 (12%) even a subtotal absence of one of the immunoglobulin fractions. 8 of the cases (14%) also showed a reduction of gammaglobulin level. In 20 children (34%), the level of one or more of the three major Ig classes was elevated. All except one became clinically normal within a few years, showing normal gamma or immunoglobulin levels when tested. The tuberculin test was positive in all patients. The length and course of the disease in these cases was similar to that in patients with normal test values. Gammaglobulin therapy did not have any beneficail effect. Consequently, it is concluded that although humoral immunological defects may be one aetiological factor, they do not play any major role in the course of chronic otitis media in children.

Effect of positioning of the feet in posturography.

Static posturography has been developed from a basic research test to a widely used clinical tool for evaluating dizzy patients. Before any actual standardization can be achieved, however, several aspects of the test situations have to be evaluated, including the position of the feet. The quantitative significance of the standing position in posturographic measurements was evaluated with healthy volunteers studied under visual and nonvisual conditions, using 4 foot positions: heels together with the toes 30 degrees apart or at an angle of the volunteer's own choice, and the feet parallel and either 0 or 10 cm apart were studied separately. Each measurement was characterized in terms of 5 parameters (body sway velocity, vibration-induced shift of centre of force in anteroposterior and lateral directions, and maximum displacement of centre of force in the same directions). Body sway velocities were smallest when the feet were parallel and 10 cm apart. Although the position chosen by the subject was usually more stable than that with the toes 30 degrees apart, the difference was nonsignificant. According to our results, the standing position is not crucial in posturographic measurements provided that the distance between the heels is determined, and the subject can just as well choose the angle between the feet if the heels are kept together.

Tumours of the parotid region.

Fifty-eight operatively treated tumours of the parotid region are presented. Of the tumours 85 per cent were benign and 15 per cent malignant. The percentage of pleomorphic adenomas was 36 per cent. During five years of observation there were no recurrences of benign tumours. Operative complications consisted of one paresis of the mandibular branch of the facial nerve and one death due to bleeding and aspiration during the first postoperative day. Frey's syndrome occurred postoperatively in 26 per cent of the cases. It is emphasized that parotid tumours should be concentrated to centres where surgeons have sufficient experience with these cases.