Molecular identification of coliform bacteria from colicky breastfed infants.

OBJECTIVE: To determine the presence of intestinal coliform bacteria in colicky vs healthy infants. STUDY DESIGN: We isolated coliform strains from faeces and performed quantitative bacterial cultures in 41 colicky and 39 healthy breastfed infants, identified using PCR with species-specific primers, strain-specific Automated Ribotyping and the API-50E kit for Enterobacteriaceae to identify the most frequent strains. RESULTS: Coliform strains were more abundant in colicky infants (median 6.04 log(10) CFU/g faeces, range 2.00-8.76) vs controls (median 4.47 log(10) CFU/g faeces, range 1.00-8.08) (p = 0.026). Escherichia coli, Klebsiella pneumoniae, K. oxytoca, Enterobacter cloacae, E. aerogenes and Enterococcus faecalis were the predominant species in colicky and healthy infants. The counts of each bacterial species differed between the two groups, and the difference was significant (p = 0.002) for E. coli: median 6.30 log(10) CFU/g faeces (range 3.00-8.74) in colicky infants, and median 4.70 log(10) CFU/g faeces (range 2.00-5.85) in controls. CONCLUSIONS: This is the first study to evaluate the colonization patterns of gas-forming coliforms in colicky infants and healthy controls identified by molecular methods. Coliform bacteria, particularly Escherichia coli, were found to be more abundant in colicky infants. Our data could help to shed light on the cause of infantile colic.

Detection of microbial concentration in ice-cream using the impedance technique.

The detection of microbial concentration, essential for safe and high quality food products, is traditionally made with the plate count technique, that is reliable, but also slow and not easily realized in the automatic form, as required for direct use in industrial machines. To this purpose, the method based on impedance measurements represents an attractive alternative since it can produce results in about 10h, instead of the 24-48h needed by standard plate counts and can be easily realized in automatic form. In this paper such a method has been experimentally studied in the case of ice-cream products. In particular, all main ice-cream compositions of real interest have been considered and no nutrient media has been used to dilute the samples. A measurement set-up has been realized using benchtop instruments for impedance measurements on samples whose bacteria concentration was independently measured by means of standard plate counts. The obtained results clearly indicate that impedance measurement represents a feasible and reliable technique to detect total microbial concentration in ice-cream, suitable to be implemented as an embedded system for industrial machines.

Growth kinetics on oligo- and polysaccharides and promising features of three antioxidative potential probiotic strains.

AIMS: To determine the antioxidative activity, glutathione production, acid and bile tolerance and carbohydrate preferences of Lactobacillus plantarum LP 1, Streptococcus thermophilus Z 57 and Bifidobacterium lactis B 933. METHODS AND RESULTS: The intact bacteria exhibited antioxidative capacity against linolenic acid and ascorbate oxidation. The antioxidative activity of cell-free extracts was determined by chemiluminescent assay and agreed with total glutathione content. Superoxide dismutase was negligible in all the strains. Bile and gastric juice resistance was tested in vitro to estimate the transit tolerance in the upper gastrointestinal tract. Bifidobacterium lactis B 933 and L. plantarum LP 1 were more acid tolerant than S. thermophilus Z 57. All the strains were resistant to bile. Among 13 indigestible carbohydrates, galacto-oligosaccharides and fructo-oligosaccharides were utilized by all the strains and did not affect survival in human gastric juice. CONCLUSIONS: These potential probiotic strains exhibited antioxidative properties and good viability in gastric juice and bile may indicate tolerance to the transit through the upper gastrointestinal tract. Galacto-oligosaccharides and fructo-oligosaccharides are the most appropriate prebiotics to be used in effective synbiotic formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: These results outline promising strains with antioxidative properties. Carbohydrate preferences can be exploited in order to develop synbiotic products.

Characterization of gram-positive broad host-range plasmids carrying a thermophilic replicon.

The cryptic plasmid pBC1 (1.6 kb) isolated from Bacillus coagulans Zu1961 was genetically marked with the genes for chloramphenicol and ampicillin resistance (CmR and ApR) from the Escherichia coli plasmid pJH101. The recombinant vector obtained (pCP49, 7.0 kb) replicated and expressed CmR in B. subtilis and CmR and ApR in E. coli. Different shuttle vectors for Gram+ bacteria were also constructed by inserting pBC1 into the Staphylococcus aureus plasmid pC194. The smallest of these, pLM6 (2.8 kb), containing essentially pBC1 and the chloramphenicol acetyl transferase gene from pC194, replicated in B. subtilis at a copy number of 60. By electroporation, these plasmids were introduced and stably maintained in B. subtilis, B. amyloliquefaciens, S. aureus, S. carnosus and Lactobacillus reuteri.

Characterization of the plasmid pMB1 from Bifidobacterium longum and its use for shuttle vector construction.

The nucleotide sequence of the 1847-bp Bifidobacterium longum B2577 cryptic plasmid pMB1 was determined. The plasmid had a G+C content of 62.0%, and contained two open reading frames, orf1 and orf2, likely arranged in an operon. The proteins encoded by orf1 and orf2 show the highest degree of similarity with similarly arranged peptide sequences translated from Corynebacterium glutamicum pXZ10142 and Mycobacterium fortuitum pAL5000 plasmids. Recombinant plasmids containing the pMB1 replicon were able to replicate in Bifidobacterium animalis MB209. The successful transformation of this strain with pMB1-based plasmids facilitated characterization of this replicon, results of which showed that both orf1 and orf2 are necessary for plasmid replication. A family of new Escherichia coli-B. animalis shuttle plasmids, based on the pMB1 replicon and expressing a cat and an ery gene, was constructed.

Effects of probiotic administration upon the composition and enzymatic activity of human fecal microbiota in patients with irritable bowel syndrome or functional diarrhea.

In a clinical trial, 10 patients suffering from irritable bowel syndrome or functional diarrhea were administered the probiotic preparation VSL-3. Preliminary results indicated that administration of VSL-3 improved the clinical picture and changed the composition and biochemistry of fecal microbiota. Titer variations of intestinal bacterial groups were evaluated by culture and PCR techniques. A significant increase in lactobacilli, bifidobacteria and Streptococcus thermophilus was observed as a consequence of probiotic treatment, while enterococci, coliforms, Bacteroides and Clostridium perfringens did not change significantly. The strains Bifidobacterium infantis Y1 and Bifidobacterium breve Y8, included in VSL-3, were specifically detected in feces of patients treated with the probiotic by using strain-specific PCR primers. In addition, fecal beta-galactosidase increased and urease activities decreased as a result of changes in the intestinal microbiota induced by VSL-3 administration.

New shuttle vector for cloning in Bacillus stearothermophilus.

Cloning vector plasmid pRP9 was constructed on the basis of the broad host-range plasmid pLM6. pRP9 was a small plasmid (2.9 kb), possessed a convenient polyrestriction site sequence and efficiently transformed Bacillus subtilis, Bacillus stearothermophilus and Escherichia coli. Furthermore, pRP9 presented a very high segregational stability in Bacillus hosts. Also, the structural stability in Bacillus strains, grown under selective pressure, of pRP9 carrying a 3-kb fragment, was high. No single-stranded and high-molecular weight pRP9 DNA was found in B. stearothermophilus. The host/vector systems described possessed all the properties required for efficient gene cloning.

Impact on the composition of the faecal flora by a new probiotic preparation: preliminary data on maintenance treatment of patients with ulcerative colitis.

BACKGROUND: Although 5-aminosalicylic acid (5-ASA) oral compounds are the standard maintenance treatment for ulcerative colitis in remission, some patients cannot use them because of side-effects. Clinical and experimental observations have suggested the potential role of probiotics in inflammatory bowel disease therapy. AIM: To evaluate the effects on intestinal microflora and the clinical efficacy of a new probiotic preparation in patients with ulcerative colitis in remission. PATIENTS AND METHODS: Twenty patients with ulcerative colitis, intolerant or allergic to 5-ASA, have been treated with a new probiotic preparation (VSL#3, CSL, Milan, Italy) containing 5x10(11) cells/g of 3 strains of bifidobacteria, 4 strains of lactobacilli and 1 strain of Streptococcus salivarius ssp. thermophilus. Two doses of 3 g were administered o.d. for 12 months. Faecal samples for stool culture were obtained from the patients at the beginning of the trial and after 10, 20, 40, 60, 75, 90 days, 12 months and at 15 days after the end of the treatment. The following bacterial groups have been evaluated in the faeces: total aerobic and anaerobic bacteria, enterococci, Streptococcus thermophilus, lactobacilli, bifidobacteria, Bacteroides, clostridia, coliforms. Patients were assessed clinically every two months, and assessed endoscopically at 6 and 12 months or in relapse. RESULTS: Faecal concentrations of Streptococcus salivarius ssp. thermophilus, lactobacilli and bifidobacteria increased significantly in all patients, compared to their basal level, from the 20th day of treatment (P<0.05) and remained stable throughout the study. Concentrations of Bacteroides, clostridia, coliforms, total aerobic and anaerobic bacteria did not change significantly during treatment (P = N.S.). Fifteen of 20 treated patients remained in remission during the study, one patient was lost to follow up, while the remaining relapsed. No significant side-effects have been reported. CONCLUSIONS: These results show that this probiotic preparation is able to colonize the intestine, and suggest that it may be useful in maintaining the remission in ulcerative colitis patients intolerant or allergic to 5-ASA. Controlled trials are warranted to confirm these preliminary results.

Genetic transformation of intact cells of Bacillus subtilis by electroporation.

Plasmid DNAs were introduced by electroporation into Bacillus subtilis PB1424 as an alternative to competent-cell or protoplast transformation. The maximum electroporation efficiency was 10(4) transformants/microgram DNA. Parameters including growth phase of cells, ionic strength of the suspending medium, concentration and size of plasmid DNAs, amplitude and duration of the pulse, were evaluated in order to determine conditions that improved transformation efficiency.

Traditional and high potency probiotic preparations for oral bacteriotherapy.

Current research continues to improve the treatment options available to clinicians for oral bacteriotherapy. Recently, a greater understanding of the role of endogenous digestive microflora has generated renewed interest in the potential of oral bacteriotherapy for the management of a wide spectrum of gastrointestinal and systemic disorders. Several treatment strategies for oral bacteriotherapy have already entered clinical trials and it is hoped that some of these strategies will become widely available in the near future. This review summarises the current status of oral probiotic preparations for bacteriotherapy and discusses any obstacles to their successful clinical development. Newer probiotic preparations include high potency preparations that are greatly enriched in lactic acid bacteria, both in terms of bacterial concentrations and the number of bacterial strains. These preparations have a greater potential for clinical effectiveness than traditional preparations and are entering clinical evaluation especially in patients with inflammatory bowel disease and pouchitis, irritable bowel syndrome, or cryptosporidiosis. The pitfalls of previous clinical investigations of traditional probiotic preparations and the perils of future clinical trials with high potency preparations are discussed in the context of unmet needs and realistic expectations of success. Although considerable progress has been made in oral bacteriotherapy, focused efforts by basic scientists and clinical investigators and continued support from pharmaceutical companies is required to successfully develop probiotics for use in clinical medicine. Newer high potency probiotic preparations appear to have a great advantage over traditional preparations and should be the area of most active biomedical research in the field.

High-performance anion-exchange chromatography coupled with pulsed amperometric detection and capillary zone electrophoresis with indirect ultra violet detection as powerful tools to evaluate prebiotic properties of fructooligosaccharides and inulin.

Fructooligosaccharides (FOS) and inulin are food grade non-digestible carbohydrates that exert beneficial nutritional effect. This paper describes the suitability of high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and capillary zone electrophoresis (CZE) to evaluate fermentation properties of FOS and inulin in pure Bifidobacterium cultures; and to study their effects on faecal cultures (microbial population and short-chain fatty acids). Prebiotic effectiveness of FOS and inulin of different degrees of polymerization was evaluated monitoring the changes in their molecular weight distribution during the in vitro growth of selected Bifidobacterium strains. The qualitative analysis of the residual soluble oligosaccharides or polysaccharides from Raftilose Synergy, Raftiline HP and Raftilose P95 was carried out by HPAEC-PAD, using a CarboPac PA 100 column and an appositely optimized gradient elution program. Under the optimized gradient elution conditions, glucose, fructose, sucrose were resolved from each other and from fructans with a DP ranging from 3 (1-kestose) to 60. The chromatographic profiles of the spent broths pointed out that almost every strain presented a different capability to ferment fructan chains of variable DP, indicating wide strain to strain differences. To explore the prebiotic effect of FOS and inulin, related to of short chain fatty acids (SCFAs) accumulation in faecal cultures due to fermentative metabolism of intestinal microflora, analysis of SCFAs, acetic and lactic acid was achieved by co-electroosmotic capillary electrophoresis, where the electrophoretic mobility of the anionic analytes and electroosmotic flow (EOF) were similarly directed. Moreover, the use of UV detection for the analyses of our organic anions required a running electrolyte which allowed indirect detection. The optimization of the capillary electrophoretic conditions was carried out by applying a chemometric study based on the use of the experimental design, the effects of three parameters, i.e. temperature, voltage and percentage of methanol added to the background electrolyte were investigated.

Specific detection of bifidobacterium strains in a pharmaceutical probiotic product and in human feces by polymerase chain reaction.

For PCR specific detection of the strains Bifidobacterium longum Y 10, B. infantis Y 1 and B. breve Y 8 used in a new probiotic product (VSL-3), strains-specific rDNA primers have been developed. Spacer regions between the 16S and 23S rRNA genes (ITS) of the three strains were amplified by PCR with conserved primers and the nucleotide sequence of these ITSs were determined. On the basis of their comparison with the rDNA sequences retrieved from GenBank, we designed new primers which specifically recognize the species B. breve and the two strains B. infantis Y 1 and B. breve Y 8. Specificity of these primers was confirmed through the analysis of 60 bifidobacteria strains belonging to the more representative human species. The feasibility of this PCR method was investigated in commercial VSL-3 product and fecal samples collected from 4 patients affected by inflammatory bowel deseases and two healthy subjects before and after the VSL-3 administration. By PCR analysis of different VSL-3 commercial batches we were successful in differentiating and quantifying the strains B. longum Y 10, B. infantis Y 1 and B. breve Y 8. B. infantis Y 1 and B. breve Y 8 could be detected at high concentration in fecal specimens of both patients and subjects treated with the probiotic preparation, showing a different colonization behaviour. Seven days after the VSL-3 treatment suspension, no patients and subjects harbored B. infantis Y 1 and B. breve Y 8, indicating a transient presence of these exogenous strains.

Technological and biological evaluation of tablets containing different strains of lactobacilli for vaginal administration.

Ten strains of lactobacilli were evaluated for the administration of viable microorganisms to restore the normal indigenous flora in the treatment of urogenital tract infections (UTI) in women. As the strains considered are facultative anaerobes, optimization of the production process was particularly critical to preserve bacterial viability. The microorganisms were formulated in single- and double-layer vaginal tablets. The two layers were characterized by different release properties: one is an effervescent composition that ensures a rapid and complete distribution of the active ingredient over the whole vaginal surface; while the second is a sustained release composition capable of releasing the lactobacilli over a longer period of time. Three different retarding polymers were tested, and all the formulations and tablets were evaluated in terms of technological processability, bacterial viability and stability, and cell adhesion properties of the microorganisms. From the results obtained, three out of ten strains appear particularly suitable for their application in the treatment of UTI. A larger batch of tablets made with a mixture of the three strains was then evaluated, confirming the feasibility of their industrial production and a good bacterial viability in the final dosage form.

Characterization of Bifidobacterium strains for use in soymilk fermentation.

Soybean milk, which serves as a base for a variety of beverages, contains raffinose, stachyose, pentanal and n-hexanal; the former two may be responsible for flatulence after fermentation, whilst the latter two for a beany flavour. Twenty-seven strains of Bifidobacterium were analyzed for their alpha-galactosidase activity and the production of lactic and acetic acids to determine their potential for use in the production of fermented soymilk. The behaviour of three strains in soymilk was studied to determine their ability to reduce alpha-D-galactosyl oligosaccharides and produce lactic and acetic acids. They all were able to reduce stachyose and raffinose. Pentanal and n-hexanal were metabolized by Bifidobacterium breve MB233. These data indicate that bifidobacteria can be used for biotechnological processes that employ soymilk as the substrate. A product with low levels of alpha-D-galactosyl oligosaccharides and alkylic aldehydes may be obtained.

Effects of rifaximin administration on the intestinal microbiota in patients with ulcerative colitis.

The effect of rifaximin on the intestinal bacterial population was studied in a clinical trial. Twelve patients with ulcerative colitis were administered rifaximin 1800 mg/day in 3 treatment periods of 10 days, each followed by 25 days of wash-out. Fecal samples were collected at the beginning and at the end of each treatment period to perform microbiological examinations. Titer variations of enterococci, coliforms, lactobacilli, bifidobacteria, Bacteroides spp., and Clostridium perfringens as well as their susceptibility to rifaximin during the different phases of the study were evaluated. The presence of Candida spp. was also monitored. After each wash-out period, concentrations of the intestinal microbial groups tested returned to initial values, showing that the administration of high doses of rifaximin does not significantly modify the colonic microbiota. Rifaximin-resistant isolates were also found, particularly in bacteria belonging to Bifidobacterium genus, included as probiotics in several fermented foods and in pharmaceutical preparations.

Regulation of dihydrodipicolinate synthase and diaminopimelate decarboxylase activity in Bacillus stearothermophilus.

The feedback inhibition of the enzymes dihydrodipicolinate (DHDPS) and diaminopimelate decarboxylase (DAPD) in the wild strain Zu 183 of Bacillus stearothermophilus and in its S-(2-aminoethyl)-cysteine resistant L-lysine overproducing strain AEC 12 was studied. The optimum temperature and pH of both enzymes were also evaluated. No inhibition of DHDPS by L-lysine, L-threonine, L-methionine and L-isoleucine was observed either in the wild strain or in the AEC 12 mutant. DAPD was completely inhibited by L-lysine and only partially by L-threonine and L-methionine in Zu 183 and AEC 12 strains, but the concentration required was found to be much higher in the AEC 12 strain. The regulation mechanism of L-lysine biosynthesis in Bacillus stearothermophilus Zu 183 was also discussed.

An embedded portable biosensor system for bacterial concentration detection.

Microbial screening is a primary concern for many products. Traditional techniques based on standard plate count (SPC) are accurate, but time consuming. Furthermore, they require a laboratory environment and qualified personnel. The impedance technique (IT) looking for changes in the electrical characteristics of the sample under test (SUT) induced by bacterial metabolism represents an interesting alternative to SPC since it is faster (3-12h vs. 24-72 h for SPC) and can be easily implemented in automatic form. With this approach, the essential parameter is the time for bacteria concentration to reach a critical threshold value (about 10(7) cfu mL(-1)) capable of inducing significant variations in the SUT impedance, measured by applying a 100 mV peak-to-peak 200 Hz sinusoidal test signal at time intervals of 5 min. The results of this work show good correlation between data obtained with the SPC approach and with impedance measurements lasting only 3h, in the case of highly contaminated samples (10(6) cfu mL(-1)). Furthermore, this work introduces a portable system for impedance measurements composed of an incubation chamber containing the SUT, a thermoregulation board to control the target temperature and an impedance measurement board. The mix of cheap electronics and fast detection time provides a useful tool for microbial screening in industrial and commercial environments.

Characterization of the superoxide dismutase SOD1 gene of Kluyveromyces marxianus L3 and improved production of SOD activity.

Superoxide dismutase (SOD) activity is one major defense line against oxidative stress for all of the aerobic organisms, and industrial production of this enzyme is highly demanded. The Cu/Zn superoxide dismutase gene (KmSOD1) of Kluyveromyces marxianus L3 was cloned and characterized. The deduced KmSod1p protein shares 86% and 71% of identity with Kluyveromyces lactis and Saccharomyces cerevisiae Sod1p, respectively. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc and in enzymatic function were conserved. To the aim of developing a microbial production of Cu/Zn superoxide dismutase, we engineered the K. marxianus L3 strain with the multicopy plasmid YG-KmSOD1 harboring the KmSOD1 gene. The production of KmSOD1p in K. marxianus L3 and K. marxianus L3 (pYG-KmSOD1) in response to different compositions of the culture medium was evaluated. The highest specific activity (472 U(SOD) mg(prot) (-1)) and the highest volumetric yield (8.8 x 10(5) U(SOD) l(-1)) were obtained by the recombinant strain overexpressing KmSOD1 in the presence of Cu(2+) and Zn(2+) supplements to the culture media. The best performing culture conditions were positively applied to a laboratory scale fed-batch process reaching a volumetric yield of 1.4 x 10(6) U(SOD) l(-1).

Urease activity in the genus Bifidobacterium.

The urease activity of 414 strains representing 21 species of the genus Bifidobacterium was surveyed. The strongest ureolytic strains belong mostly to the species B. suis and only a few to B. breve, B. magnum and "subtile" homology group. The study of some strongly ureolytic strains showed that urea and organic nitrogen concentration did not influence urease production. The high urease activity found also in the absence of urea suggested that this enzyme is not inducible. An ammonia concentration of 14 mM did not repress urease activity.

Amino acids produced by bifidobacteria and some Clostridia.

A lot of 121 strains of bifidobacteria and 9 strains of clostridia were examined for their ability to release free amino acids in the culture broth. The bifidobacteria studied belong to 18 species or "homology group" and the clostridia to 8 species. The growth in a synthetic medium with ammonium salts as sole nitrogen source was also studied. All the clostridia and the majority of the bifidobacteria produce various amino acids. The possible ecological significance of these findings is suggested.