In vitro inhibitory activity of probiotic spore-forming bacilli against genotoxins.

AIMS: To investigate the ability of bacilli of various species (Bacillus clausii, Bacillus subtilis, Bacillus lentus, Bacillus pumilus. Bacillus megaterium, Bacillus firmus, Bacillus sp.) and origins (probiotic and collection strains) to counteract the activity of some representative DNA-reactive agents. METHODS AND RESULTS: The inhibitory effect of 21 bacilli strains, previously characterized by tDNA-PCR, on four genotoxins, was examined in vitro using the short-term assay SOS-Chromotest. All strains had a high inhibitory activity against 4-nitroquinoline-1-oxide and N-methyl-N'-nitro-nitrosoguanidine (direct agents), whereas the inhibitory activity was high or moderate against 2-amino-3,4-dimethylimidazo[4,5-f]quinoline and aflatoxin B1 (indirect agents). Antigenotoxicity was observed in vegetative cells, but not heat-treated cells or spore suspensions. The spectroscopic properties of compounds were modified after cell co-incubation and all the strains maintained high viability after exposure to the genotoxins. CONCLUSIONS: No relevant differences in antigenotoxicity were evidenced among strains of the examined species or between probiotic and collection strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Although derived from an in vitro model, the results suggest that Bacillus-based probiotics could be useful for reducing the gastrointestinal risk originating from genotoxic agents.

Tolerance to challenges miming gastrointestinal transit by spores and vegetative cells of Bacillus clausii.

AIMS: To study Bacillus clausii from a pharmaceutical product (Enterogermina O/C, N/R, SIN, T) and reference strains (B. clausii and Bacillus subtilis) for eco-physiological aspects regarding the gut environment. METHODS AND RESULTS: Spores and vegetative cells were challenged in vitro miming the injury of gastrointestinal transit: pH variations, exposure to conjugated and free bile salts, microaerophilic and anaerobic growth. No relevant differences were found studying the growth at pH 8 and 10, whereas at pH 7 the yields obtained for O/C and SIN were higher than those obtained for N/R and T strains. The spores were able to germinate and grow in the presence of conjugated bile salts (up to 1%, w/v) or free bile salts (0.2%) and also exhibited tolerance for the combined acid-bile challenge. As evidenced by lag-time, growth rate and cell yield the tolerance of Enterogermina isolates for conjugated salts was comparable with that of B. clausii type strain (DSM 8716(T)), and resulted higher than that observed for B. subtilis (ATCC 6051(T)). All the considered B. clausii strains demonstrated microaerophilic growth, but only some grew anaerobically in a nitrate medium. CONCLUSIONS: The ability of B. clausii spores to germinate after an acid challenge and grow as vegetative cells both in the presence of bile and under limited oxygen availability is consistent with the beneficial health effects evidenced for spore-forming probiotics in recent clinical studies. SIGNIFICANCE AND IMPACT OF THE STUDY: The experimental evidence from this study emphasizes some functional properties of B. clausii strains regarding their use as probiotics.

Screening of potential lactobacilli antigenotoxicity by microbial and mammalian cell-based tests.

Antigenotoxicity is considered an important property for probiotic lactobacilli. The ability of non probiotic lactobacilli from dairy products and starters to inhibit two reference genotoxins: 4-nitroquinoline-1-oxide and N-methyl-N'-nitro-N-nitrosoguanidine was evaluated. The study was carried out using short-term assays with different targets, such as procaryotic cells (SOS-Chromotest for genotoxicity in Escherichia coli and Ames test for mutagenicity in Salmonella typhimurium) and eucaryotic cells (Comet assay for genotoxicity in Caco-2 enterocytes). A high proportion of strains inhibiting 4-nitroquinoline-1-oxide activity was found in Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus plantarum. Inhibition of N-methyl-N'-nitro-N-nitrosoguanidine activity occurred in only one L. acidophilus strain. All the strains with antigenotoxic properties also demonstrated antimutagenic activity and produced modifications in genotoxin spectroscopic profiles. Strain viability during and after genotoxin exposure was confirmed. Concordance of the results obtained with microbial and mammalian cell-based tests is underlined.

[A new conservative approach in the treatment of postoperative digestive-tract fistulas. Mechanical closure by a balloon-catheter]. / Nuova metodica conservativa nel trattamento delle fistole digestive postoperatorie. Chiusura meccanica con catetere con palloncino.

BACKGROUND: Digestive fistulas represent troublesome complication in patients operated in modern surgical wards where the improved surgical procedures and better intensive care enhance the surgeon to perform more aggressive approaches with a high surgical risk index. The management of a patient presenting a digestive-tract fistula is never easy, being its approach either conservative (TPN) or surgical. We applied an alternative surgical procedure consisting in a mechanical closure of the fistula using a balloon-catheter so as to improve outcome in those patients in whom medical tratment did not show satisfactory RESULTS. METHODS: We treated 7 patients presenting a postoperative fistula following several surgical procedures for neoplasms of the digestive system. These fistulas were closed using a Foley or Fogarthy balloon catheter preceeded by radiological and/or endoscopy controls. Once the catheter was placed, oral nutrition was started and some patients were discharged. A progressive deflation of the balloon was performed until complete removal of the catheter upon approx 10 days. RESULTS: We obtained a complete healing of the fistula in 6 patients, within 10 days since catheter placement. Only one patient required another operation. CONCLUSIONS: Our case-series may seem statistically not significant, but varied concerning location and type of fistulas. We observed an excellent outcome using this procedure which allows very short healing period thanks to an early oral nutrition uptake and a decrease in costs mainly due to a short hospital stay and a minor use of expensive drugs (TPN).

Bacterial amylolytic activity enhances beta-glucuronidase expression of amylase-negative Escherichia coli strain in starch-medium.

The possibility of associating starch degradation with bacterial beta-glucuronidase expression was examined. We proved that starving, in starch medium, amylase-negative Escherichia coli (M94) which has constitutive beta-glucuronidase greatly reduces (p < 0.01) its background activity, but the addition of both cell-free supernatants or cells of Bacillus subtilis (B10) producing amylase greatly increases (p < 0.01) the E. coli beta-glucuronidase activity. Increases in activity were maximal when amylase in the medium ranged from 0.3 to 0.8 U ml-1 and pH from 6.8 to 6.3, whereas higher amylase activity interacted with E. coli viability and the effect on beta-glucuronidase was less evident. The impact of B. subtilis amylase on E. coli beta-glucuronidase induction, observed when the organisms were co-cultured, indirectly supports the hypothesis that amylolytic activity of hindgut bacteria may be effective on beta-glucuronidase induction of the climax microflora. This last finding is important in the health field, considering the implication between the deconjugating role of this enzyme and consequent activation of toxic and carcinogenic compounds.

Implications of alpha-amylase production and beta-glucuronidase expression in Escherichia coli strains.

Two Escherichia coli strains in which alpha-amylase production differed were used to study in depth some characteristics related to beta-glucuronidase induction by starch. The beta-glucuronidase background activity in Luria broth medium was comparable for the two isolates, but only amylase positive S1 was able to grow on starch molecules supplied as the sole carbon source. In this case growth resulted at higher beta-glucuronidase levels (p < 0.01) with respect to basal activity and the induced expression was maximal (6.1-fold) when cultures reached the stationary phase. Growth in the presence of a protein synthesis inhibitor (chloramphenicol) was associated with a marked reduction of activity. The beta-glucuronidase activity of amylase negative M94 remained unchanged during starvation on starch medium, but an induced response was observed with methylumbelliferyl-glucuronide. These results further support the hypothesis that starch metabolism is involved in the complex beta-glucuronidase regulation of E. coli strains. This is relevant not only for basic research but also to investigating gut microbial enzymology.

Effect of different starches on Escherichia coli (S1) beta-glucuronidase expression.

Studies with pure cultures growing in laboratory media indicated that beta-glucuronidase expression of Escherichia coli S1 was considerably affected by starch added to the medium as the only carbon source. This result, which may be an aspecific modulation of enzyme expression, was independent of the starch molecular structure and effects were analogous for maize, rice, wheat or potato starches. It was observed that enzyme expression was little affected by the growth rate. The beta-glucuronidase activities of starch-grown bacteria found in the present study agree with those observed in animal and human models performed for in vivo evaluation of effects of dietary starch effects on gut microbial ecosystems.

High Prevalence of Vibrio cholerae Non-O1 Carrying Heat-Stable-Enterotoxin-Encoding Genes among Vibrio Isolates from a Temperate-Climate River Basin of Central Italy.

Vibrio spp. of clinical interest from the Arno River basin (Tuscany, Italy) were investigated in this study. Vibrios were isolated from 70% of water samples. Vibrio cholerae non-O1 was the most prevalent species (82% of isolates), followed by Vibrio mimicus (10%) and Vibrio metschnikovii (8%). Recovery of vibrios was correlated with temperature, pH, and various indicators of municipal pollution. None of the 150 Vibrio isolates carried ctx-related genomic sequences, whereas 18 (14.6%) of the 123 V. cholerae non-O1 isolates and 1 (6.7%) of the 15 V. mimicus isolates carried sto alleles. These findings indicate that considerable circulation of sto-positive vibrios may occur in temperate-climate freshwater environments.

Comparison of fluorogenic and conventional membrane filter media for enumerating coliform bacteria.

4-Methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl-beta-D-glucuronide were added to MacConkey broth and their diagnostic powers for total coliforms (TC) and Escherichia coli, respectively, were tested by membrane filtration at primary isolation. Examining water samples from different sources proved the usefulness of fluorogenic rather than reference media both as regards recovery efficiency and rapidity (possible within 12 h) of analyses. The recoveries obtained by fluorogenic and conventional tests for both TC and E. coli were correlated. Values were comparable in surface water samples, while a higher sensitivity of fluorogenic media was observed in samples of shallow contaminated ground water. Results seem to indicate that the use of fluorogenic membrane filtration analysis for colimetric indicators could be favourably considered especially for sanitary surveying of drinking water.

Carbohydrate enriched diets and bacterial glycosidases in rat faeces.

The expression of some faecal hydrolytic enzymes in rats fed for 4 months on sucrose or starch enriched diets was compared with a standard diet. The assay reliability was confirmed for animal and experimental variability. The beta-D-glucuronidase and beta-D-glucosidase activities were always higher in rats fed on starch than on other diets. N-acetyl-beta-D-glucosaminidase and N-acetyl-beta-D-galactosaminidase showed decreased activity when passing from a standard to a sucrose, and from a sucrose to a starch diet. There was little modification in the levels of faecal alpha-D-glucosidase, sulphatase and protease with the various experimental diets.

beta-N-acetylhexosaminidases from Serratia marcescens.

Two forms of beta-N-acetylhexosaminidase from Serratia marcescens with an optimum pH of 5.0 and 6.5, respectively, to 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside were separated by DEAE-cellulose chromatography and Sephacryl S-200 chromatography. On the basis of their molecular weights, thermal stability, substrate specificity and isoelectric points, the form with an acidic pH optimum resembled hexosaminidase B, whereas the form with a neutral pH optimum resembled hexosaminidase C. Lectin binding studies showed that the acidic form does not bind to concanavalin-A-Sepharose, Tetragonolobus purpurea-agarose, wheat germ-agglutinin-Sepharose or Ricinus communis-agglutinin-agarose, whereas the neutral form binds to the last two lectin columns.

Fluorogenic detection of atypical coliforms from water samples.

We examined the effectiveness of fluorogen in detecting bacterial enzymes in atypical or injured coliform strains in environmental water samples. 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl-beta-D-glucuronide, substrates for beta-galactosidase and beta-glucuronidase respectively, were used as markers for total and faecal coliform bacteria and it was confirmed that fluorogenic assays have a greater sensitivity than reference methods. It was also observed that adding MU-conjugates (50 micrograms/ml) to low selective media for membrane filtration, besides shortening test times, reduces false negative results when detecting sanitary microbial indicators of water pollution.

[Development of antibiotic resistance in purified sewage effluents subjected to chlorination]. / Evoluzione dell'antibiotico-resistenza in effluenti fognali depurati sottoposti a clorazione.

Antibiotic-resistance is widely spread phenomenon in the environment because of uncontrolled discharge of urban and animal wastewaters. Sewage treatment can significantly reduce the number of both sensitive and resistant bacteria. A reduction of about 1.5 logarithmic units in faecal coliforms was observed during biological treatment (3, 7), but a simultaneous increase in the percentage of resistant strains occurred because of not well understood selection phenomena. The above reported bacterial reduction is not always sufficient to meet the quality standards of Italian legislation required to discharge the treated effluents into surface waters, and so, chlorination become a compulsory additional treatment whose impact on both sensitive and resistant microflora must be evaluated. The results obtained in the present research have demonstrated that chlorine concentrations in the range of 0.5-2 ppm are able to reduce significantly the faecal coliforms concentrations and, in particular, treatment with 1 ppm of chlorine for 1 hour reduces the concentration of the above reported bacteria to the extent of 2 logarithmic units, so that their final concentration are of the about 10(2)/100 ml. The surviving chlorine tolerant bacteria seem to be antibiotic resistant in higher percentage than the chlorine sensitive ones and so, as a consequence, a significant increase in the antibiotic resistance and multiresistance was observed in the chlorinated effluents. In this context it is interesting to underline the larger variety of resistance patterns observed in the chlorine-resistant bacteria in comparison with the uniformity in the resistance patterns observed in isolated from unchlorinated effluents. The selected chlorine-tolerant strains seem to be less able to transfer their resistances under laboratory conditions, not because of curing effect of chlorine on the plasmids but, probably, because of the damage to cellular cell envelopes.

The effect of anaerobic and aerobic wastewater treatment on faecal coliforms and antibiotic-resistant faecal coliforms.

The efficiency of anaerobic treatment of animal wastes and aerobic treatment of urban sewage in removing faecal coliforms and their effect on the antibiotic-resistant coliforms was evaluated in this study. A two reactor anaerobic digester and six activated sludge plants were studied. The concentrations of both faecal coliforms in sampling from influents and treated effluents were calculated to determine efficiency of plants during depuration treatments. Anaerobic and aerobic treatments resulted in 90% and 97% (arithmetic mean values) efficiency in removing faecal coliforms. Although neither anaerobic nor aerobic treatment seems to significantly increase the percentage of antibiotic-resistant bacteria, during the aerobic treatment of urban sewage there was a tendency for the percent of antibiotic-resistant bacteria to increase.

[Submerged-phase gel electrophoresis in the analysis of plasmids for epidemiologic purposes]. / Gel elettroforesi in fase sommersa nell'analisi plasmidica a fini epidemiologici.

The validity of the submarine agarose gel electrophoresis as routine method for plasmidic epidemiology was considered. Using standard plasmids, the efficiency of a performed protocol conditions (5 V/cm for 3 hours, 0.8% agarose) in screening plasmids of different size was studied. The results provide sharp profile resolution and an accurate estimate of molecular weights in the range from 1.2 to 112 megadaltons.

Resistotype heterogeneity of Escherichia coli strains isolated from humans, animals and sewage waters.

The tests commonly used for bacterial identification, especially in the field of microbial environmental analyses frequently do not provide a sufficient strain differentiation. Considering the importance that accurate characterization of bacterial pollution indicators could have as epidemiological tools, this study used a resistogram subtyping method for Escherichia coli as a tentative method of effecting a good monitoring of the environmental spread of this microorganism. The resistance of 313 E. coli strains of different origin (human, animal, sewage), previously identified by standard biochemical reactions, to 8 chemical compounds (inorganic, organic and dyes) and to 7 antibiotics was tested. The results indicated this method has a higher discriminatory power for chemicals than for drugs. Some typical resistotype patterns for E. coli from various sources are described.

Relationship between environment spread and presence in hosts of Escherichia coli strains resistant to antibiotics and metals.

The antibiotic and metal resistance percentages of 315 E. coli strains, isolated from: a sample population not-exposed to antibiotics, hospitalized inpatients and from bred animals, were compared with the resistance percentages of 217 environmental isolates (sewage and river isolates). The highest levels of resistance and multiresistance were found for clinical and river isolates. A wider number of resistance markers, observed for environment isolates with respect to human and animal isolates, made it possible to hypothesis e that the resistant strains could possess some selective advantage that enhances their survival in the environment. Association of antibiotic resistance and metal-resistance has been demonstrated in all isolates but it is particularly evident in the environment and clinical isolates. Correlation analysis revealed that the patterns of antibiotic and metal resistance of environmental E. coli isolates are in good agreement with those of human origin and that sewage and river E. coli isolates are well correlated too.

Binding of interleukin 2 to gangliosides.

Exogenous gangliosides inhibit interleukin 2 (IL2)-dependent growth of a T cell line, AKIL -1.E8. IL2 activity is retained by columns of ganglioside covalently linked to poly(L-lysine)-agarose and is not eluted with ethylene glycol but is completely recovered by elution with 1% SDS. The ability of gangliosides to inhibit IL2 activity is directly related to the complexity of their carbohydrate portion, and related ceramide derivatives at similar concentrations do not inhibit IL2 activity. We conclude that IL2 bound to exogenous gangliosides is inactive and that the carbohydrate portion of the ganglioside is crucial to its interaction with IL2.