Biomedical subjects

W E Sandine

Publications and source records attributed to W E Sandine.

At least 19 recordsLinked to original sources

PubMed1997

The ldh phylogeny for environmental isolates of Lactococcus lactis is consistent with rRNA genotypes but not with phenotypes.

Lactate dehydrogenase (ldh) gene sequences, levels of 16S rRNA group-specific probe binding, and phenotypic characteristics were compared for 45 environmental isolates and four commercial starter strains of Lactococcus lactis to identify evolutionary groups best suited to cheddar cheese manufacture, ldh sequences from the environmental isolates showed high similarity to those from two groups of L. lactis used for industrial fermentations, L. lactis subsp. cremoris and subsp. lactis. Within each phylogenetically defined subspecies, ldh sequence similarities were greater than 99.1%. Strains with phenotypic traits formerly diagnostic for both subspecies were found in each ldh similarity group, but only strains belonging to L. lactis subsp. cremoris by both the newer, genetic and the older, superseded phenotypic criteria were judged potentially suitable for the commercial production of cheddar cheese. Identical evolutionary relationships were inferred from ldh sequences and from binding of subspecies-specific, 16S rRNA-directed oligonucleotide probes. However, groups defined according to these chromosomal traits bore no relationship to patterns of arginine deamination, carbon substrate utilization, or bacteriophage sensitivity, which may be encoded by cryptic genes or sexually transmissible genetic elements. Fourteen new L. lactis subsp. cremoris isolates were identified as suitable candidates for cheddar cheese manufacture, and 10 of these were completely resistant to three different batteries of commercial bacteriophages known to reduce starter activity.

Bacterial Typing Techniques↗

PubMed1997

Expression of ropy and mucoid phenotypes in Lactococcus lactis.

Strains of Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris were cultured under aerobic and anaerobic conditions on plates of whey agar, Elliker agar, and M17L agar at 15, 20, and 30 degrees C to determine the environmental conditions required for the expression of the ropy phenotype. Two strains, L. lactis ssp. cremoris Ropy 352 and L. lactis ssp. cremoris Hollandicus, exhibited two distinct polysaccharide phenotypes, ropy and mucoid. Expression of these phenotypes could be induced individually or simultaneously. The inducible nature of this response suggests that genetic regulators were present. Western blots were used to determine whether or not Lon protease and RcsA, two regulators of polysaccharide expression in Escherichia coli, were present in lactococci. Lon, a negative regulator, and RcsA, an unstable positive regulator, have been shown at the structural level to be conserved in a number of Gram-negative and Gram-positive microorganisms. The present study found evidence for structural conservation of Lon protease in lactococci. Less of the Lon-like protein was observed in the ropy strains than in the nonropy strains.

ATP-Dependent Proteases↗

PubMed1996

Characterization and sequence analysis of a stable cryptic plasmid from Enterococcus faecium 226 and development of a stable cloning vector.

A small cryptic plasmid, pMBB1, isolated from Enterococcus faecium 226 was characterized. The plasmid contained an extremely stable replicon which has limited homology to the lactococcal plasmid pCI305. Sequence analysis of the replicon detected one open reading frame of 822 bp capable of encoding a 32-kDa protein. No detectable single-stranded intermediates were found for the replicon, suggesting that pMBB1 may be included in the same family as pCI305, although pCI305 exhibits a more narrow host range. A small stably maintained vector able to replicate in a variety of lactic acid bacteria, containing a large multiple cloning region, was constructed by using the pMBB1 replicon.

Amino Acid Sequence↗

PubMed1996

Biomodulation of the toxic and nutritional effects of small bowel bacterial overgrowth in end-stage kidney disease using freeze-dried Lactobacillus acidophilus.

Small bowel bacterial overgrowth (SBBO), well known to occur in end-stage kidney failure, is responsible for producing uremic toxins and contributing to the patient's decreased nutritional well-being. In this study, 8 hemodialysis patients were treated with a course of oral Lactobacillus acidophilus (LBA) in an attempt to alter this SBBO. LBA treatment was effective in lowering 2 compounds generated in vivo. Serum dimethylamine (DMA) levels dropped from 224 +/- 47 to 154 +/- 47 micrograms/dl at the end of LBA treatment (p < 0.001). Nitrosodimethylamine, a carcinogen, levels also decreased significantly from 178 +/- 67 (untreated) to 83 +/- 49 ng/kg (after LBA treatment). Patients nutritional status, assessed as serum albumin, body weight, caloric intake, midarm muscle area (MAMA) and appetite improved modestly, but not significantly. LBA changed small bowel pathobiology by modifying metabolic actions of SBBO, reducing in vivo generation of toxins and carcinogens and promoting nutrition with no adverse side effects.

Analysis of Variance↗

PubMed1995

Characteristics of Tn5307 exchange and intergeneric transfer of genes associated with nisin production.

Transfer of the Lactococcus lactis 11454 nisin-sucrose conjugative transposon, Tn5307, was investigated to develop a methodology for conjugation of this element to other lactic acid bacteria. Tn5307 exchange was sensitive to temperature and pH but was not affected by protease or amylase treatments to donor cells. Moreover, conjugation studies demonstrated that the direct-plate method could be employed to rapidly identify LM2301 transconjugants able to transfer Tn5307 at least ten times more efficiently than 11454. Intergeneric transfer of nisin and sucrose genes between L. lactis and a dairy Enterococcus sp. was also investigated. Erythromycin-resistant Enterococcus sp. recipients were developed by electro-transformation with pGK13 or by conjugal introduction of the broad-host-range plasmid pAM beta 1. Matings between L. lactis 11454 and an Enterococcus sp. recipient that contained pAM beta 1 yielded sucrose-positive, nisin-immune transconjugants at a frequency of 2.3 x 10(-9) transconjugants per donor cfu. Agar-overlay assays for nisin production revealed that enterococcal transconjugants did not produce the bacteriocin, but DNA.DNA hybridization with a nisA-specific probe demonstrated that these bacteria had acquired the nisin structural gene.

Anti-Bacterial Agents↗

PubMed1995

A milk-based method for detecting antimicrobial substances produced by lactic acid bacteria.

A new technique for the detection of antimicrobial substances produced by lactic acid bacteria has been developed. In this technique, milk agar plates were supplemented with tetrazolium chloride or tetrazolium blue dyes. Comparisons of milk agar assays with M17 agar plates indicated that, out of 30 bacterial strains, 13 strains produced bacteriocins or inhibitory substances that were detectable on milk agar plates but not on M17 agar plates. Multiple-strain lactococcal cultures are used in milk fermentations. To identify suitable strains to combine for industrial use, component strains must be tested for compatibility. The procedure described allows optimization of compatibility. The assay of putative producer and sensitive indicator strains using milk agar plates (11% nonfat dry milk plus .8% agar and .02% tetrazolium chloride or tetrazolium blue) provides an important tool to prevent allopathic interactions in mixed cultures.

Agar↗

PubMed1995

Polysaccharide expression in Lactococci.

Lactococcus lactis produces polysaccharide under defined environmental conditions. Three approaches are being used to identify regulators of polysaccharide synthesis in the organism. i) Two new lactococcal vectors, each of which contains a promoterless reporter gene, have been developed. They are being used to select for chromosomal insertions that affect expression of polysaccharide ii) Genetic complementation with lactococcal genomic libraries identified two classes of lactococcal genomic activities, both of which affect capsule expression in Escherichia coli iii) Lon, a highly conserved protease, is a negative regulator of polysaccharide synthesis in E. coli. Antiserum specific to Lon protease reacted with a lactococcal protein similar in size to E. coli Lon. This reaction was strongest in lactococcal strains which could not be induced to express polysaccharide.

Cloning, Molecular↗

PubMed1994

Quaternary ammonium compounds in milk: detection by reverse-phase high performance liquid chromatography and their effect on starter growth.

A reverse-phase HPLC method is described that permits detection of quaternary ammonium sanitizers in milk. The commercial mixture of quaternary ammonium sanitizer used, n-alkyl (50% C14, 40% C12, and 10% C16) dimethyl benzyl ammonium chloride, was extracted from raw and homogenized milks by the Mojonnier milk fat extraction protocol, selectively eluted from the reverse-phase column in the presence of organic solvent and ion-pairing agent, and then analyzed by diode array detection at 217 and 280 nm. Quaternary ammonium sanitizer concentrations in augmented milk samples in the range of 1.0 to 60 micrograms/ml were analyzed with high precision as indicated by a small variance around the mean. Starter culture strains currently in use for manufacture of various fermented milk products available in the Department of Microbiology Culture Collection were examined for their sensitivity to quaternary ammonium sanitizer. All strains were greatly inhibited by as little as 20 micrograms/ml in milk; some were inhibited by only 10 micrograms/ml.

Animals↗

PubMed1993

Methods to demonstrate the bactericidal activity of bacteriocins.

Two simple techniques were developed to demonstrate bactericidal activity of bacteriocins. Both were based on allowing a lawn of indicator strain to grow first, then exposing the lawn to bacteriocin-containing cell-free supernatants in a well cut in the seeded agar lawn or by inoculating the bacteriocin-producing strain onto the indicator lawn. Lysis of cells of the indicator strain resulted in a clear zone. These techniques may be adapted to test antimicrobial substances other than bacteriocins and to help to determine their modes of action.

Bacteriocins↗

PubMed1993

Isolation of Lactococcus lactis subsp. cremoris from nature by colony hybridization with rRNA probes.

Lactococcus lactis subsp. cremoris is widely used in the manufacture of fermented milk products. Despite numerous attempts, efforts to isolate new strains by traditional plating and identification methods have not been successful. Previously, we described oligonucleotide probes for 16S rRNAs which could be used to discriminate L. lactis subsp. cremoris from related strains. These probes were used in colony hybridization experiments to screen large numbers of colonies obtained from enrichment cultures. A total of 170 strains of L. lactis were isolated from six milk samples, two colostrum samples, and one corn sample by using oligonucleotide probe 212RLa specific for the species L. lactis. Fifty-nine of these isolates also hybridized to L. lactis subsp. cremoris-specific probe 68RCa, and 26 of the strains which hybridized to the L. lactis subsp. cremoris-specific probe had the L. lactis subsp. cremoris phenotype.

Animals↗

PubMed1993

Development and use of a selective medium for isolation of Leuconostoc spp. from vegetables and dairy products.

A selective medium (LUSM medium) for the isolation of Leuconostoc spp. was developed. This medium contained 1.0% glucose, 1.0% Bacto Peptone (Difco), 0.5% yeast extract (BBL), 0.5% meat extract (Difco), 0.25% gelatin (Difco), 0.5% calcium lactate, 0.05% sorbic acid, 75 ppm of sodium azide (Sigma), 0.25% sodium acetate, 0.1% (vol/vol) Tween 80, 15% tomato juice, 30 micrograms of vancomycin (Sigma) per ml, 0.20 microgram of tetracycline (Serva) per ml, 0.5 mg of cysteine hydrochloride per ml, and 1.5% agar (Difco). LUSM medium was used successfully for isolation and enumeration of Leuconostoc spp. in dairy products and vegetables. Of 116 colony isolates obtained from fresh raw milk, curdled milk, or various vegetables, 115 were identified as members of the genus Leuconostoc. A total of 89 of these isolates were identified to species; 13.5% of the isolates were Leuconostoc cremoris, 7.9% were Leuconostoc mesenteroides subsp. mesenteroides, 11.2% were Leuconostoc mesenteroides subsp. dextranicum, 16.9% were Leuconostoc mesenteroides subsp. paramesenteroides, 10.1% were leuconostoc lactis, and 40.4% were Leuconostoc oenos. When we compared the counts obtained for two Leuconostoc strains, Leuconostoc dextranicum 181 and L. cremoris JLL8, on MRS agar and LUSM medium, we found no significant difference between the values obtained on the two media.

Bacteriological Techniques↗

PubMed1991

A membrane protein is required for bacteriophage c2 infection of Lactococcus lactis subsp. lactis C2.

Phage-resistant mutants, isolated from cultures of Lactococcus lactis subsp. lactis C2 infected with phage c2, did not form plaques but bound phage normally. The mutants were sensitive to another phage, sk1, although the number of plaques was reduced approximately 56% and the plaques were four times smaller. Binding to phage sk1 was reduced about 10%. Another group of phage-resistant mutants, isolated from cultures infected with phage sk1, bound normally to both phages c2 and sk1 but did not form plaques with either phage. Carbohydrate analyses by gas chromatography of the cell walls showed no significant differences in saccharide compositions between the wild-type and phage-resistant cells. However, a difference was observed in the interactions of the phage with the cytoplasmic membranes. Membranes from the wild-type cells, but not mutant cells, inactivated phage c2. Phage sk1 was not inactivated by membrane from either strain. Treatment of wild-type membranes with proteinase K eliminated the ability of the membrane to inactivate the phage, whereas treatment with mutanolysin had no effect. On the basis of this ability to inactivate the phage, a membrane protein was partially purified by gel filtration and ion-exchange chromatography. Under nondenaturing conditions, the phage-inactivating protein has an apparent Mr of approximately 350,000. The protein has an apparent subunit size of 32 kDa, which suggests that it normally exists as a multimer with 10 to 12 subunits or in association with other membrane components. It is proposed that this protein is required for phage c2 infection.

Bacterial Proteins↗

PubMed1991

Antimicrobial activity of Microgard against food spoilage and pathogenic microorganisms.

Microgard, a commercially available fermented milk product containing antimicrobial metabolites, was a potent inhibitor for Gram-negative bacteria such as Pseudomonas, Salmonella, and Yersinia when 1% concentration was incorporated into agar media. Gram-positive Bacillus cereus, Staphylococcus aureus, and Listeria monocytogenes were insensitive to Microgard. Kluyveromyces marxianus, an unidentified black yeast, and Penicillium expansum were partially suppressed, whereas Aspergillus niger and a yogurt spoilage yeast were tolerant to 5% Microgard. Optimum activity of Microgard was at pH 5.3 and below; the concentration that gave complete inhibition depended upon the number of bacteria present as well as the genus tested. Blood agar base reversed the antagonistic activity of Microgard against Pseudomonas putida compared with plate count agar.

Anti-Bacterial Agents↗

PubMed1990

Highly selective medium for isolation of Listeria monocytogenes from food.

A new selective medium (Al-Zoreky-Sandine listeria medium [ASLM]) was formulated to recover Listeria monocytogenes from food specimens; the medium completely inhibited common food microflora. Recognition of Listeria colonies is evident by black discoloration of the medium due to esculin hydrolysis without need for special illuminating equipment. The medium contains acriflavin, ceftazidime, and moxalactam as selective agents. Compared with Listeria Selective Agar, ASLM was equally effective in recovering L. monocytogenes. However, ASLM inhibited micrococci, enterococci, and gram-negative bacteria, especially a strain that mimicked L. monocytogenes on Listeria Selective Agar. The new medium was able to recover heat injured cells with only 15% less count than the nonselective medium.

Bacteria↗

PubMed1990

The bacteriophage kh receptor of Lactococcus lactis subsp. cremoris KH is the rhamnose of the extracellular wall polysaccharide.

A receptor for bacteriophages of lactic acid bacteria, including Lactococcus lactis subsp. cremoris KH, was found on the cell wall and not on the cell membrane, as determined by a phage-binding assay of sodium dodecyl sulfate- and mutanolysin-treated cell walls. The cell wall carbohydrates of L. lactis subsp. cremoris KH were analyzed by gas chromatography and mass spectrometry and found to contain rhamnose, galactose, glucose and N-acetylglucosamine. Similar analysis of mutants that were reduced in the ability to bind phages kh, 643, c2, ml3, and 1 indicated that galactose was essential for binding all phages. In addition, rhamnose was required for binding phages kh and ml3. Inhibition studies of phage binding by using two different lectins with a specificity for galactose indicated that phage kh may not bind directly to galactose. Rather, galactose may be an essential structural component located in the vicinity of the receptor. Incubation of any of the five phages with rhamnose or of phage kh with purified cell walls inactivated the phages. Inactivation required divalent cations and was irreversible. Inactivation of phages was stereospecific for rhamnose, as neither L-(+)- nor D-(-)-fucose (the stereoisomers of rhamnose) inhibited the phage. Furthermore, phage infection of a culture was completely inhibited by the addition of rhamnose to the medium. Therefore, the receptor for phage kh appears to be a rhamnose component of the extracellular wall polysaccharide.

Bacteriophages↗

PubMed1989

Lactobacillus effects on cholesterol: in vitro and in vivo results.

A double blind investigation was conducted on the influence of a commercially available tablet containing Lactobacillus acidophilus and Lactobacillus bulgaricus (Lactinex Becton Dickinson Microbiology Systems, Cockeysville, MD) on human serum lipoprotein concentrations. Tablets containing about 2 X 10(6) viable bacteria of Lactobacillus mixtures or placebo tablets were ingested by 354 nonfasting informed subjects in a dose of one tablet each, taken four times a day. There was a 3-wk washout period between two 6-wk treatment periods. The number of viable lactobacillus in unused returned tablets was the same at the end of the study as in the beginning. Analysis of paired data using Wilcoxon signed ranks test showed no major effects on lipoprotein concentrations for either the placebo-treated group or the lactobacilli-treated group. There were no statistically significant differences for low density lipoprotein concentrations between the lactobacilli-treated group and the placebo-treated group. The high density lipoprotein concentrations increased 1.8 to 3.0 mg/dl in both groups for both study periods. For total cholesterol the placebo-treated group experienced a statistically significant increase in the first period according to the Wilcoxon signed ranks test (from 208.0 to 215.0 mg/dl, P less than .001) but not according to a two-sample Student t test. Total cholesterol did not change significantly for the Lactobacillus-treated group in either period. Cardiac risk factor (ratio of total cholesterol to high density cholesterol) did not vary during the study. Lipoprotein values increased immediately following vigorous exercise compared with following 15 min of resting without either placebo or treatment. Sample controls for assay and reassay gave virtually identical values (coefficient of variation 1.6%), confirming that assay results were quite reliable. Thus, ingestion of commercially available Lactobacillus tablets, which contain about 2 X 10(6) cfu/tablet of L. acidophilus and L. bulgaricus cells in a dose of four tablets daily did not affect serum lipoprotein concentrations.

Buffers↗

PubMed1988

New nomenclature of the non-rod-shaped lactic acid bacteria.

Bacteria of the Lactococcus (formerly lactic Streptococcus) genus are described to emphasize certain less well-known facts of importance in their successful use as dairy starters. Other streptococci (non-rod forms) are briefly characterized to emphasize their usefulness in industrial milk fermentations.

Animals↗

PubMed1988

Inhibitory action of nisin against Listeria monocytogenes.

The sensitivity of nine strains of Listeria to nisin was determined as well as the minimum inhibitory concentration of nisin necessary to completely inhibit growth of these strains. All strains tested were variably sensitive to nisin and different MIC values were obtained, ranging from 740 to 10(5) IU/ml in trypticase soy agar and from 1.85 to 10(3) IU/ml in MRS agar. The inhibition of L. monocytogenes ATCC 7644 in TSB trypticase at different pH values and in sterilized and nonsterilized cottage cheese by nisin (37 X 10(2) IU/ml and 2.55 X 10(3) IU/g, respectively) also was investigated. This bacterium was completely inhibited after 24 h at pH 5.0 and above. At pH 4.5, 4.0, and 3.5, it was inhibited within 24 h. In cottage cheese no Listeria survivors were found at 24 h at 37 and 4 degrees C whether or not the cheese had been sterilized when as many as .35 X 10(6) cell/g were added at zero time.

Cheese↗