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Phylogeny of the defined murine microbiota: altered Schaedler flora.

The "altered Schaedler flora" (ASF) was developed for colonizing germfree rodents with a standardized microbiota. The purpose of this study was to identify each of the eight ASF strains by 16S rRNA sequence analysis. Three strains were previously identified as Lactobacillus acidophilus (strain ASF 360), Lactobacillus salivarius (strain ASF 361), and Bacteroides distasonis (strain ASF 519) based on phenotypic criteria. 16S rRNA analysis indicated that each of the strains differed from its presumptive identity. The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains of Lactobacillus murinis and Lactobacillus animalis (both isolated from mice), and all of these strains probably belong to a single species. Strain ASF 360 is a novel lactobacillus that clusters with L. acidophilus and Lactobacillus lactis. Strain ASF 519 falls into an unnamed genus containing [Bacteroides] distasonis, [Bacteroides] merdae, [Bacteroides] forsythus, and CDC group DF-3. This unnamed genus is in the Cytophaga-Flavobacterium-Bacteroides phylum and is most closely related to the genus Porphyromonas. The spiral-shaped strain, strain ASF 457, is in the Flexistipes phylum and exhibits sequence identity with rodent isolates of Robertson. The remaining four ASF strains, which are extremely oxygen-sensitive fusiform bacteria, group phylogenetically with the low-G+C-content gram-positive bacteria (Firmicutes, Bacillus-Clostridium group). ASF 356, ASF 492, and ASF 502 fall into Clostridium cluster XIV of Collins et al. Morphologically, ASF 492 resembles members of this cluster, Roseburia cecicola, and Eubacterium plexicaudatum. The 16S rRNA sequence of ASF 492 is identical to that of E. plexicaudatum. Since the type strain and other viable original isolates of E. plexicaudatum have been lost, strain ASF 492 is a candidate for a neotype strain. Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any organisms whose 16S rRNA sequences are currently in the GenBank database. The 16S rRNA sequence information determined in the present study should allow rapid identification of ASF strains and should permit detailed analysis of the interactions of ASF organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms.

Animals↗

Evaluation of two self-care treatments for prevention of vaginal candidiasis in women with HIV.

Vaginal candidiasis (VC) is a common concern for women living with HIV infection. The authors evaluated the effectiveness of two self-care approaches to prophylaxis of VC among HIV-infected women, weekly intravaginal application of Lactobacillus acidophilus or weekly intravaginal application of clotrimazole tablets, in a randomized, double-blind, placebo-controlled trial. VC was defined as a vaginal swab positive for Candida species in the presence of signs/symptoms of vaginitis and the absence of a diagnosis of Trichomonas vaginalis or bacterial vaginosis. Thirty-four episodes of VC occurred among 164 women followed for a median of 21 months. The relative risk of experiencing an episode of VC was 0.4 (95% CI = 0.2, 0.9) in the clotrimazole arm and 0.5 (95% CI = 0.2, 1.1) in the Lactobacillus acidophilus arm. The estimated median time to first episode VC was longer for clotrimazole (p = .03, log rank test) and Lactobacillus acidophilus (p = .09, log rank test) compared with placebo. Vaginal yeast infections can be prevented with local therapy. Education about self-care for prophylaxis of VC should be offered to HIV-infected women.

Administration, Intravaginal↗

Effects of Lactobacillus strains on cancer cell proliferation and oxidative stress in vitro.

AIMS: The objective of this study was to assess in vitro, whether heat-killed (HK) lactic acid bacteria cells and fractionations of HK cells could suppress the viability of human cancer cells and inhibit the cytotoxicity associated with oxidative stress. METHODS AND RESULTS: Among the strains, the HK cells of Lactobacillus acidophilus 606 and Lactobacillus casei ATCC 393 exhibited the most profound inhibitory activity in all of the tested cell lines. HK cells of L. acidophilus 606 were determined to be less toxic to healthy human embryo fibroblasts (hEF cells) than were HK cells of L. casei ATCC 393. The soluble polysaccharides from L. acidophilus 606 evidenced the most effective anticancer activity, but inhibited hEF cell growth by only 20%. The soluble polysaccharides from L. acidophilus 606 were partly observed to induce apoptosis in the HT-29 cells by DNA fragmentation and propidium iodine staining. Both the HK cells of L. acidophilus 606 and the soluble polysaccharide components of this strain also exhibited potent antioxidative activity. CONCLUSIONS: Our findings suggest that the soluble polysaccharide fraction from L. acidophilus 606 may constitute a novel anticancer agent, which manifests a high degree of selectivity for human cancer cells and antioxidative agent in the food industry. SIGNIFICANCE AND IMPACT OF THE STUDY: These soluble polysaccharide components from Lactobacillus may be applied to various foods, and used as adjuncts for cancer therapy and prevention.

Antineoplastic Agents↗

Andean yacon root (Smallanthus sonchifolius Poepp. Endl) fructooligosaccharides as a potential novel source of prebiotics.

The ability of three known probiotic strains (two lactobacilli and one bifidobacterium) to ferment fructooligosaccharides (FOS) from yacon roots (Smallanthus sonchifolius Poepp. Endl) was compared to commercial FOS in this study. Results indicate that Lactobacillus acidophilus NRRL-1910, Lactobacillus plantarum NRRL B-4496, and Bifidobacterium bifidum ATCC 15696 were able to ferment yacon root FOS. FOS consumption apparently depended on the degree of polymerization and the initial FOS composition. L. plantarum NRRL B-4496 and L. acidophilus NRRL B-1910 completely utilized 1-kestose molecules, while B. bifidum was able to utilize 1-kestose molecules as well as molecules with a higher degree of polymerization.

Asteraceae↗

Influence of nonfermented dairy products containing bacterial starter cultures on lactose maldigestion in humans.

The effect of nonfermented dairy products containing yogurt or acidophilus cultures on lactose utilization by lactose-maldigesting humans was investigated. Yogurt and acidophilus milk containing 10(7) or 10(8) of Streptococcus thermophilus and Lactobacillus bulgaricus, or Lactobacillus acidophilus, respectively, were prepared using commercially processed 2% low fat milk. Immediately following inoculation, products were refrigerated. Lactose maldigestion was monitored by measuring breath hydrogen excretion at hourly intervals for 8 h following consumption of 400 ml of each test meal containing approximately 20 g of lactose. The yogurt milk containing 10(8) cfu/ml was shown to contain significant concentrations of microbial beta-galactosidase (EC 3.2.1.23; approximately 3 U/ml), which remained stable for at least 14 d at refrigerator temperatures. Breath hydrogen peaks were delayed and significantly lower (approximately 20 ppm at 5 to 7 h) than control values (approximately 70 ppm at 4 h), and intolerance symptoms were eliminated in all subjects. Yogurt milk containing 10(7) cfu/ml demonstrated intermediate breath hydrogen values and was marginally significantly different from control values. Lactobacillus acidophilus strains with varying resistance to bile and total beta-galactosidase-producing potential were also tested. Only one strain, LA-1, which demonstrated low bile resistance and intermediate beta-galactosidase activity, was capable of significantly decreasing breath hydrogen values when 10(8) cfu/ml of milk was consumed.

Adult↗

[Isolation, assay and biotyping of lactobacillary flora of the vagina].

The results of the isolation, quantitation and biotyping of 120 strains of vaginal Lactobacillus are presented. The more effective culture media for isolation and different methods of pH measurement were investigated. Five different species of Lactobacillus were isolated in vaginal specimens (Lactobacillus acidophilus 88.8%). The size limits found in the 120 Lactobacillus acidophilus strains were 0.8 to 6,0 mu length and 0.3 to 0.8 mu width. The effort to correlate number of Lactobacillus to pH and weight of vaginal fluid did not give satisfactory results because of difficulties in collecting homogeneous samples. Biochemical investigation of 120 vaginal strains of Lactobacillus did not show any peculiar biotype. The A.A. suggested the "routine" detection of vaginal lactobacillar flora as a usefull marker of ecological status in the choice of the therapy.

Adolescent↗

Detection and identification of Lactobacillus species in crops of broilers of different ages by using PCR-denaturing gradient gel electrophoresis and amplified ribosomal DNA restriction analysis.

The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 10(8) to 10(9) CFU per gram of crop contents. Many of the lactobacilli present in the crop (61.9% of isolates) belonged to species of the Lactobacillus acidophilus group and could not be differentiated by PCR-DGGE. A rapid and simple ARDRA method was developed to distinguish between the members of the L. acidophilus group. HaeIII-ARDRA was used for preliminary identification of isolates in the L. acidophilus group and to identify Lactobacillus reuteri and Lactobacillus salivarius. MseI-ARDRA generated unique patterns for all species of the L. acidophilus group, identifying Lactobacillus crispatus, Lactobacillus johnsonii, and Lactobacillus gallinarum among crop isolates. The results of our study provide comprehensive knowledge of the Lactobacillus microflora in the crops of birds of different ages using nucleic acid-based methods of detection and identification based on current taxonomic criteria.

Aging↗

The formation of germtubes by Candida albicans, when grown with Staphylococcus pyogene, Escherichia coli, Klebsiella pneumoniae, Lactobacilius acidophilus and Proteus vulgaris.

The formation of germtubes by twelve clinical isolates of C. albicans was studied in human serum containing per millilitre 10(3) to 10(9) organisms as: Staphylococcus pyegene, Escherichia coli, Klebsiella pneumoniae, Lactobacillus acidophilus and Proteus vulgaris. All the five bacteria inhibited formation of germtubes by C. albicans at all concentrations and the percent germtube formed diminished with increasing concentration of the bacteria. Lactobacillus acidophilus inhibited the formation of germtubes maximally followed by Staphylococcus pyogene, Escherichia coli and Klebsiella pneumoniae. Proteus vulgaris in the concentrations of 10(3) to 10(7) bacteria per millilitre produced only insignificant inhibition of formation of germtubes by C. albicans. Since germtubes of C. albicans are invasive, it is suggested that inhibition of "blastospore-germtube transformation" may be significantly responsible for prevention of infection by C. albicans by coexisting bacterial flora.

Candida albicans↗

Effect of various types of fermentation on in vitro protein and starch digestibility of differently processed pearl millet.

Pearl millet (Pennisetum typhoideum) grains were fermented with Lactobacilli and yeast alone, in combination and with natural flora at 30 degrees C for 48 h after giving various processing treatments viz, fine and coarse grinding, soaking, debranning, dry heat treatment, germination and autoclaving after adding of water. Fermentation was carried out with Lactobacillus acidophilus and Rhodotorula isolated from naturally fermented pearl millet and Lactobacillus acidophilus, Candida utilis and natural fermentation using freshly ground pearl millet flour as inoculum. All the processing treatments except coarse grinding improved the protein and starch digestibility. Autoclaving enhanced the digestibilities of processed samples which was further improved by different types of fermentation, the maximum being in case of germinated and naturally fermented pearl millet. A combination of Lactobacilli and yeast was more effective in increasing the protein as well as starch digestibility as compared to pure culture fermentation.

Fermentation↗

Effect of Lactobacillus inoculation and antibiotic feeding of chickens on availability of dietary biotin.

Oral inoculation with Lactobacillus acidophilus and dietary supplementation with antibiotics were compared in broiler chicks fed various levels of biotin to determine their effect on the biotin status of the chicks. Lactobacillus acidophilus inoculation of broiler chickens fed a diet marginally deficient in biotin (56 micrograms/kg) caused a significant decrease (P less than .01) in their liver biotin content from 1596 to 931 ng/g. The decrease biotin status was confirmed by significant increases (P less than .01) in 16:1 and 18:1 plasma fatty acids and significant decreases (P less than .01) in 18:0 and 20:4 omega 6 plasma fatty acids. These alterations in fatty acids were only observed when the samples were taken from chickens in the fed condition. Supplementation with Lactobacillus caused an increased incidence of foot dermatitis and decreased growth of chickens, presumably by competing with the chicken for dietary biotin. No significant changes were noted with antibiotic feeding.

Animals↗

[Mycoplasmas and dysplasia of the uterine cervix].

The Authors report a case-list of 395 patients vaginal specimens who were never treated with chemo-antibiotic therapy. Cell dysplastic impairments were found in 213 cases. About these dysplastic alterations, 133 are of slight type, 53 intermediate type and 27 are in advanced phase. We can say, about the last 27 cases, that the concomitance of pH greater than 6.1 in 67% of the cases and the absence of Lactobacillus acidophilus in 81.4% of the cases is not casual. Furthermore, we can notice that vaginal pH suffers an increase in dysplastic patients with a smaller colonization with Lactobacillus acidophilus that, in dysplastic advanced phase is absent in 81.4% of the cases. It is also to remark a significant increase of cases Trichomonas-positive and Mycoplasmas-positive in dysplastic patients, as compared with normal women. The results of the case-list, even if preliminary, seem to be indicative for an evolution of the studies on the relationship between uterine cervix cells and Mycoplasmas and eventual possibility the Mycoplasmas can act as carriers of oncogenic viruses such as Herpes and Papova Virus.

Animals↗

Expression of a heterologous manganese superoxide dismutase gene in intestinal lactobacilli provides protection against hydrogen peroxide toxicity.

In living organisms, exposure to oxygen provokes oxidative stress. A widespread mechanism for protection against oxidative stress is provided by the antioxidant enzymes: superoxide dismutases (SODs) and hydroperoxidases. Generally, these enzymes are not present in Lactobacillus spp. In this study, we examined the potential advantages of providing a heterologous SOD to some of the intestinal lactobacilli. Thus, the gene encoding the manganese-containing SOD (sodA) was cloned from Streptococcus thermophilus AO54 and expressed in four intestinal lactobacilli. A 1.2-kb PCR product containing the sodA gene was cloned into the shuttle vector pTRK563, to yield pSodA, which was functionally expressed and complemented an Escherichia coli strain deficient in Mn and FeSODs. The plasmid, pSodA, was subsequently introduced and expressed in Lactobacillus gasseri NCK334, Lactobacillus johnsonii NCK89, Lactobacillus acidophilus NCK56, and Lactobacillus reuteri NCK932. Molecular and biochemical analyses confirmed the presence of the gene (sodA) and the expression of an active gene product (MnSOD) in these strains of lactobacilli. The specific activities of MnSOD were 6.7, 3.8, 5.8, and 60.7 U/mg of protein for L. gasseri, L. johnsonii, L. acidophilus, and L. reuteri, respectively. The expression of S. thermophilus MnSOD in L. gasseri and L. acidophilus provided protection against hydrogen peroxide stress. The data show that MnSOD protects cells against hydrogen peroxide by removing O(2)(.-) and preventing the redox cycling of iron. To our best knowledge, this is the first report of a sodA from S. thermophilus being expressed in other lactic acid bacteria.

Cloning, Molecular↗

Reduction of fecal shedding of enterohemorrhagic Escherichia coli O157:H7 in lambs by feeding microbial feed supplement.

Enterohemorrhagic Escherichia coli O157:H7, an emerging food-borne pathogen, has been implicated in several outbreaks in the US. Ruminants, including cattle, sheep and deer are reservoirs of E. coli O157:H7 and fecal shedding of the pathogen forms the vehicle of entry into the human food chain. We studied the efficacy of Lactobacillus acidophilus, Streptococcus faecium, a mixture of L. acidophilus and S. faecium and a mixture of L. acidophilus, S. faecium, Lactobacillus casei, Lactobacillus fermentum and Lactobacillus plantarum in reducing fecal shedding of E. coli O157:H7 by sheep experimentally infected with the pathogen prior to administration with the microbials. Following oral inoculation with 10(10)CFU of E. coli O157:H7, 30 Suffolk ram lambs were blocked by body weight (six blocks of five lambs each) and lambs within the block randomly assigned to five groups. The lamb groups were fed daily for 7 weeks a basal diet without microbial supplement (control) or the basal diet with L. acidophilus or with S. faecium or with a mixture of L. acidophilus and S. faecium or with a mixture of L. acidophilus, S. faecium, L. casei, L. fermentum and L. plantarum. The microbial supplements contained stabilized live naturally occurring bacteria and were mixed with the diet at the rate of 6.0x10(6)CFU per kilogram of diet. Fecal samples were collected weekly and analyzed for E. coli O157:H7 using modified tryptic soy broth with novobiocin as a pre-enrichement broth and cefixim-tellurite sorbitol MacConkey agar (CT-SMAC) as a selective media. E. coli O157:H7 was confirmed by its reaction with O157 and H7 antisera. E. coli O157:H7 was shed continuously and in varying numbers in the feces throughout the 7-week experimental period by all five groups. However, lambs administered a mixture of L. acidophilus, S. faecium, L. casei, L. fermentum and L. plantarum shed significantly lower (P=0.0211) average number of E. coli O157:H7 (2.3log(10)CFU per gram of feces per week) than the other lamb groups over the entire experimental period. S. faecium supplemented lambs were comparable (P=0.0884) to lambs fed a mixture of L. acidophulus and S. faecium in fecal shedding of the pathogen (3.5 versus 4.4log(10)CFU per gram of feces) but significantly lower (P=0.0001) than the control lambs (5.6log(10)CFU per gram of feces) and those supplemented with L. acidophilus (5.5log(10)CFU per gram of feces). Average daily gain (ADG) and gain to feed ratio (G:F) were significantly improved (P=0.0145) by the mixed culture microbials (163.0g and 0.33 for the control, 186.4g and 0.37 for L. acidophulus, 168.2g and 0.36 for S. faecium, 213.6g and 0.46 for L. acidophulus and S. faecium, and 219.1g and 0.44, respectively for L. acidophilus, S. faecium, L. casei, L. fermentum and L. plantarum supplemented lambs. The study indicates that supplementing lambs infected with E. coli O157:H7 with S. faecium or a mixture of S. faecium, L. acidophilus, L. casei, L. fermentum and L. plantarum in the diet can reduce total number of E. coli O157:H7 shed in the feces and improve animal meat production performance as well.

Journal Article↗

Bile salt deconjugation and cholesterol removal from media by Lactobacillus casei.

Lactobacillus casei N19 and E5 and Lactobacillus acidophilus L1 and ATCC 43121 were compared for their ability to deconjugate bile salts and remove cholesterol from MRS broth during growth at pH 6.0 and during growth without pH control. Samples grown without pH control dropped to pH 4.2 to 4.5 during 20 h of incubation, depending on the culture. The plate counts indicated that populations in all cultures were near their maximum numbers after 16 h of growth. The amount of cholesterol removed from the broth was similar for both strains of L. acidophilus grown with and without pH control. However, the strains of L. casei differed significantly in the amount of cholesterol removed during growth with or without pH control. Both cultures of L. casei that were grown at pH 6.0 removed very little cholesterol from the broth, but cells grown without pH control removed up to 60 micrograms of cholesterol/ml. All cultures of both species deconjugated 60 to 90% of the bile salts. Lactobacillus acidophilus L1 was the only culture to demonstrate differences between the two pH treatments in the amount of bile salts deconjugated; however, there was no difference in the amounts of cholesterol removed. These results indicate that most of the cholesterol removal from broth by L. acidophilus was due to assimilation, perhaps by the incorporation of cholesterol into the cellular membrane. Lactobacillus casei most likely removes cholesterol from broth by means of the destabilization of cholesterol micelles and the coprecipitation of the cholesterol with the deconjugated bile salts at pH less than 6.0.

Bile Acids and Salts↗

Oral bacterial therapy promotes recovery from acute diarrhea in children.

Lactobacilli in the intestines play an important role in developing natural defenses against both intestinal bacterial and viral infections. So a prospective clinical study was carried out at Cathay General Hospital to determine the effect of Lactobacillus acidophilus and Bifidobacterium infantis on the course of acute diarrhea in hospitalized children. Altogether 100 children between 6 and 60 months of age were collected and randomly allocated into 2 groups. Study group (n = 50) was given Infloran Berna, which contains 10(9) viable Lactobacillus acidophilus and 10(9) Bifidobacterium infantis, one capsule tid for 4 days and control group (n = 50) received parenteral rehydration only without any medication. Only 20 stool cultures in study group had positive culture results for Lactobacillus or Bifidobacterium. All children were evaluated for the degree of dehydration before rehydration. The clinical course of diarrhea was followed during the treatment period. Features on admission were similar between the study group and control group in age, duration of diarrhea at home, serum sodium & potassium and dehydration degree. The duration of diarrhea was defined as the time until the last appearance of watery stool. There was no difference between the study group and control group in the frequency of diarrhea stools on the day before admission (p > 0.05). However, the frequency of diarrhea for study group improved on the first and second day of hospitalization with statistical difference (p < 0.01). The duration of diarrhea during hospitalization in study group also decreased (3.1 vs. 3.6 days, p < 0.01). Oral bacterial therapy is an effective adjuvant therapy in rotavirus positive and negative children with diarrhea and can safely be administered during an episode of acute diarrhea.

Acute Disease↗

[Clinical assessment of a new oral bacterial treatment for children with acute diarrhea].

This study was carried out to evaluate the efficacy of a new oral bacteriotherapeutic drug in 63 children under 4 years old affected by infective diarrhoea or by extraintestinal infective pathology caused by oral antibiotic therapy. The patients were divided into three groups: the first and the second included subjects affected by acute diarrhoea enrolled in a comparative and controlled study; the third included subjects in chemotherapeutic treatment because suffering from infective extraintestinal pathologies, these patients were enrolled in a not controlled study. The first group underwent the rehydratant treatment for the acute enteritis together with the therapy with inactivated Lactobacillus acidophilus LB to value its antidiarrheic activity. To the second group (control group) was administered a no bacteriotherapeutic drug, while to the third group was administered the antibiotic therapy for extra-intestinal pathologies together with inactivated Lactobacillus acidophilus to avoid possible phenomena due to intestinal dysmicrobism. For all the patients included in the study, were reported; the number, the characteristics, the consistency of the motions and the description of the symptomatology during the treatment and daily. The greater part of the patients affected by the intestinal infective pathology subdued bacteriologic tests by coproculture. The obtained results showed a statistically significant clinical improvement of the first group patients compared with those of the second group, while those of the third group showed that the drug activity prevents the intestinal dysmicrobism affections due to the antibiotic therapy.

Acute Disease↗

Antibiotic-induced D-lactic acidosis.

OBJECTIVE: To describe a case of oral antibiotic-induced D-lactic acidosis in a patient with enteric overgrowth of Lactobacillus acidophilus. DESIGN: Single case study. SETTING: University-affiliated community hospital. INTERVENTION: Oral carbohydrate challenge test with 4000 kcal/d. MAIN RESULTS: A patient had several episodes of D-lactic acidosis after receiving oral antibiotics. Stool cultures yielded Lactobacillus acidophilus resistant to the implicated agents. Provocative challenge with dietary carbohydrate alone, in the absence of antibiotics, failed to reproduce the syndrome. CONCLUSIONS: Oral antibiotics may induce D-lactic acidosis in patients with the short-bowel syndrome by promoting the overgrowth of resistant D-lactate-producing organisms. Interactions between carbohydrate intake and antibiotic use are likely determinants in the development of this syndrome. Periodic use of stool cultures with antimicrobial susceptibility testing may assist in the management of these patients by optimizing the selection of antimicrobial agents.

Acidosis, Lactic↗

Conservation of the major cold shock protein in lactic acid bacteria.

Primers designed from consensus regions of the major cold shock gene of different bacterial species were used in PCR amplification of Lactic Acid Bacteria (LAB). An appropriately-sized PCR product was obtained from Lactococcus lactis subsp. lactis LL43-1 and MG1363; Lactococcus lactis subsp. cremoris LC10-1, LC11-1, and LC12-1; Streptococcus thermophilus ST1-1; Enterococcus faecalis EF1-1; Lactobacillus acidophilus LA1-1; Lactobacillus helveticus LH1-1; Pediococcus pentosaceus PP1-1; and Bifidobacterium animalis BA1-1. The PCR products were cloned and sequenced. The deduced amino acid sequences displayed high sequence similarity with the major cold shock proteins of Escherichia coli and Bacillus subtilis and the human Y-box factor. The amino acid residues of the cold shock domain implicated in nucleic acid binding in several unrelated species were also highly conserved in the LAB strains. It is possible, therefore, that this protein in LAB may also act as a transcriptional enhancer to other cold shock genes and/or act as an RNA chaperone unwinding tightly folded RNA molecules.

Amino Acid Sequence↗