Bacteriocin production by Streptococcus thermophilus in complex growth media.

OBJECTIVE: To test if the production of bacteriocins by Streptococcus thermophilus is influenced when grown in various complex media commonly used for the culturing of lactic acid bacteria. RESULTS: Forty-one strains of S. thermophilus were screened for the production of bacteriocins in tryptone/yeast extract/lactose (TYL), M17-lactose (M17L), M17-glucose (M17G) and MRS media. Two strains, ST144 and ST145, were identified as novel bacteriocin producers, with constitutive production observed only in M17G. Strains ST110, ST114 and ST134 constitutively produced bacteriocins in all growth media but ST114 required growth in MRS for its antimicrobial activity to persist in a 24 h culture. The addition of a synthetic quorum sensing peptide (BlpC) induced bacteriocin production by ST106 in all media tested; and by ST118 in TYL and M17L. Strain ST109, which constitutively produced a bacteriocin in TYL and M17 broths, required BlpC induction when grown in MRS. Real-time PCR analysis showed that the natural expression of blpC in ST109 was lower when grown in MRS, suggesting that something in medium interfered with the blp quorum sensing system. CONCLUSION: As the choice of growth medium influences both bacteriocin production and peptide stability, several types of production media should be tested when screening for novel bacteriocin-producing strains of S. thermophilus.

Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110.

γ-aminobutyric acid (GABA) is generated from glutamate by the action of glutamic acid decarboxylase (GAD) and characterized by hypotensive, diuretic, and tranquilizing effects in humans and animals. The production of GABA by lactic acid starter bacteria would enhance the functionality of fermented dairy foods including cheeses and yogurt. The survey of 42 strains of the yogurt starter culture Streptococcus thermophilus by PCR techniques indicated the presence of a glutamate decarboxylase gene (gadB) in 16 strains. DNA sequencing data indicated that the GAD/GABA antiporter locus (gadB/gadC) in GAD(+) S. thermophilus strains is flanked by transposase elements (5' and 3') and positioned between the luxS (5') and the HD-superfamily hydrolase genes (3'). The PCR amplification product of a ca. 2-kb genomic fragment that included the gadB and its putative promoter region was inserted into a shuttle vector, which was used to transform Escherichia coli DH5α. Subsequently, the recombinant plasmid pMEU5a-1/gadB (7.24 kb) was electrotransformed into the GAD-negative strain S. thermophilus ST128. The ST128 transformants carrying the plasmid-encoded gadB produced functional GAD enzyme as evidenced by the conversion of glutamate to GABA at a rate similar to strains with the gadB/gadC operon located on the chromosome. The results demonstrated the potential to impart to non-GABA-producing strains of S. thermophilus and other lactic acid bacteria the GAD(+) phenotype that improves their appeal in possible applications in the development of health-promoting functional foods.

Percutaneous approaches to mitral valve regurgitation.

Mitral regurgitation is a common problem associated with significant morbidity and mortality. Mitral valve surgery has been the treatment of choice for symptomatic patients with severe mitral regurgitation or asymptomatic patients with high-risk clinical features. However, a significant number of patients remain untreated related mainly due to a projected high surgical risk. Therefore, alternative percutaneous treatments including indirect annuloplasty, which takes advantage of the coronary sinus, and direct annuloplasty have recently been explored. Most recently, promising results of the first randomized trial comparing conventional mitral valve surgery to percutaneous therapy with a clip creating a double orifice much like the surgical Alfieri approach have been presented. Finally, percutaneous mitral valve replacement in an animal model has been pursued. This review serves to familiarize the reader with some anatomical concepts and devices for percutaneous mitral repair.

Emergence of the concept of platelet reactivity monitoring of response to thienopyridines.

Clinical trials have demonstrated the beneficial impact of clopidogrel in preventing major adverse cardiovascular events (MACE), particularly in patients undergoing percutaneous coronary intervention (PCI). The concept of biological clopidogrel resistance emerged with the finding of persistent platelet activation despite clopidogrel therapy in some patients. Further, a link between biological clopidogrel resistance and thrombotic recurrence after PCI was observed and a threshold of platelet reactivity (PR) for thrombotic events was suggested. Consistently, in recent trials, enhanced PR inhibition translated into a reduction in the rate of MACE after PCI. This review aims to present the emergence of the concept of PR monitoring in patients undergoing PCI following recent advances in this field.

Drug-eluting stent thrombosis.

When compared to bare metal stents (BMS), drug-eluting stents (DES) are associated with a dramatic reduction in restenosis and target lesion revascularization. However, the benefit of DES is limited to restenosis, and DES utilization does not translate into reductions in death or myocardial infarction. Additionally, concern exists regarding the long-term safety of DES, as there appears to be a small but real increase in late (LST) and very late stent thrombosis (VLST), seen particularly after the discontinuation of antiplatelet therapy. The specter of LST and VLST has curtailed enthusiasm for widespread DES utilization mandating critical appraisal of DES and the optimal role they play in percutaneous coronary intervention. The incidence of DES thrombosis is debated and varies somewhat by definition. The mechanisms are multifactorial, and involve patient, lesion, stent and physician related factors. Some of these factors are modifiable at the physician-patient level, while others are not. This review focuses on DES thrombosis, with particular attention paid to the definitions, incidence, mechanisms and clinical implications.

Molecular organization of plasmid pER13 in Streptococcus thermophilus.

Molecular features of the 4139-bp plasmid pER13 found in the dairy fermentation bacterium Streptococcus thermophilus ST113 include five open reading frames (ORFs). ORF1, ORF2 and ORF3 encode proteins for transcriptional repression (CopG), replication (RepB) and mobilization (Mob) that share homology with corresponding proteins of the pMV158 plasmid family, while ORF4 and ORF5 encode putative proteins with unspecified functions. Sequence homologies shared with plasmids found in group B and group D streptococci imply the possibility for genetic exchange with the food-grade S. thermophilus. The structural features of pER13 may be useful in designing strategies for gene transfer in lactic fermentation bacteria.

Pediocin production by recombinant lactic acid bacteria.

Production of the anti-listerial bacteriocin, pediocin, by lactic acid bacteria (LAB) transformed with the cloning vector pPC418 (Ped+, 9.1 kb) was influenced by composition of media and incubation temperature. Maximum pediocin production, tested against Listeria innocua, by electrotransformants of Lactococcus lactis ssp. lactis was measured in tryptone/lactose/yeast extract medium after 24 h growth at 30 degrees C, while incubation at 40 degrees C was optimum for Ped+ transformants of Streptococcus thermophilus and Enterococcus faecalis. The amount of pediocin produced by S. thermophilus in skim milk and cheese whey supplemented with 0.5% yeast extract was estimated as 51,000 units ml(-1) and 25,000 units ml(-1), respectively. Pediocin production remained essentially unchanged in reconstituted skim milk or whey media diluted up to 10-fold. The results demonstrate the capacity of recombinant strains of LAB to produce pediocin in a variety of growth media including skim milk and inexpensive cheese whey-based media, requiring minimum nutritional supplementation.

Promoter activity of the pER341-borne ST(Phsp) in heterologous gene expression in Escherichia coli and Streptococcus thermophilus(1).

A 138-bp EcoO109/HinfI fragment of Streptococcus thermophilus plasmid pER341 (2798 bp) including the promoter sequence of the heat stress protein gene hsp16.4 was tested in vector constructs for ability to activate the promoterless green fluorescent protein gene (gfp) from a jelly fish in Escherichia coli, S. thermophilus, and lactococci. ST(Phsp) promoted gfp expression in transformed hosts as evidenced by the presence of green fluorescent (GFP(+)) colonies under UV illumination. The results confirmed the potential of ST(Phsp) as a functional promoter in heterologous gene expression in dairy fermentation bacteria.

Distribution of plasmid-borne stress protein genes in Streptococcus thermophilus and other lactic acid bacteria.

The presence of heat stress protein genes (hsp) was tested by Southern hybridization analysis in total DNA extracts from species of the genus Streptococcus (47 strains), Lactobacillus (34 strains), Lactococcus (24 strains), and Leuconostoc (5 strains). The biotinylated hsp16.4 probe prepared from an ORF2 fragment of pER341 (2.8 kb) tested positively with restricted DNA extracts of seven Streptococcus thermophilus strains and a single strain of Lactococcus lactis subsp. cremoris. In all positive S. thermophilus strains, the hsp was located on plasmids ranging from ca. 2.8 kb to 11 kb in size, while hsp was present in a 7.5-kb plasmid in Lactococcus lactis subsp. cremoris. Southern blots with a rep probe showed that all hsp16.4+ plasmids in S. thermophilus strains also shared homology with the replication function (rep) of pER341, suggesting the common origin of these plasmids.

Molecular characterization of the erythromycin resistance plasmid pPV142 from Staphylococcus simulans.

The 2.5-kb erythromycin resistance (EmR) plasmid pPV142 of Staphylococcus simulans 13044 was isolated and characterized. Sequence analysis identified ORF1 and ORF2 encoding a 158-residue replication protein (Rep142) and a 244-residue erythromycin resistance protein (Erm, rRNA adenine N-6-methyltransferase), respectively. Structural analysis and Southern hybridization showed that the rep and ermM genes in pPV142 shared homology with the EmR plasmid pPV141 (2.4 kb) of S. chromogenes 3688 and other EmR plasmids known to exist in staphylococci and bacilli. Based on the presence of a 61-bp repeat upstream of the ermM gene, pPV142 is apparently a unique member of the pSN2 family of EmR plasmid able to express erythromycin resistance constitutively.

Structural and functional properties of the hsp16.4-bearing plasmid pER341 in Streptococcus thermophilus.

The plasmid pER341 (2798 bp) of Streptococcus thermophilus ST134 was sequenced and its open reading frame (ORF) regions were characterized. Analysis of nucleotide sequences showed the putative translation product of ORF1 (rep) sharing a high level of homology with replication proteins of several small plasmids present in lactic acid bacteria and staphylococci. This and homology of regions of plus-strand (ORI) and minus-strand (ssoA) origin of replication with pC194-class plasmids indicated that pER341 replicates by the rolling-circle mechanism. ORF2 corresponded to a putative hsp gene that apparently encodes Hsp16.4, a 142-amino-acid heat stress protein. Hsp16.4 shared significant identity with other small, 18-kDa-class heat stress proteins from prokaryotic and eukaryotic sources. Hsp16.4 is apparently the first plasmidborne low-molecular-weight heat stress protein reported in dairy fermentation bacteria with a potential role in temperature-regulated functions in S. thermophilus.

Permeabilization of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus with ethanol.

Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus cultures were treated with ethanol and tested for viability and beta-galactosidase activity. Exposure of the biomass of test cultures to 30%-55% ethanol (vol/vol) caused a 100% loss of viability and up to 15-fold increase in measurable beta-galactosidase activity in both streptococci and lactobacilli. Ethanol-treated cell suspensions could be stored for up to 6 months without loss of enzyme activity. The nonviable permeabilized biomass of the more active S. thermophilus was used to achieve up to 80% hydrolysis of lactose in aqueous solutions and non-fat milk.

Molecular properties of the erythromycin resistance plasmid pPV141 from Staphylococcus chromogenes.

The 2.3-kb erythromycin resistance (EmR) plasmid pPV141 of Staphylococcus chromogenes 3688 was isolated and characterized. Nucleotide sequence analysis identified ORF1 and ORF2 separated by a 445-bp spacing, encoding a 158-residue replication protein (Rep141) and a 244-residue erythromycin resistance protein (Erm, rRNA adenine N-6-methyltransferase), respectively. Structural analysis and Southern hybridization showed that the rep and ermM genes in pPV141 shared homology with other known EmR plasmids. Based on sequence analysis, pPV141 was classified as a unique member of the pSN2 family of EmR plasmids.

Diastolic dysfunction of the left ventricle. A review of the physiology, causes, diagnosis, treatment and implications.

Diastolic dysfunction of the left ventricle frequently occurs in people with left ventricular hypertrophy and coronary artery disease. It is a common cause of congestive heart failure, especially in the elderly. The mechanism of diastolic dysfunction, its causes, diagnosis and treatment, are reviewed. These are important factors to a Medical Director who must assess the results of non-invasive studies. Alerted by the possibility of diastolic dysfunction, the Medical Director can be more sensitive to other signs and symptoms that may represent early signs of congestive heart failure or ischemia.

Native promoter-plasmid vector system for heterologous cholesterol oxidase synthesis in Streptococcus thermophilus.

The cholesterol oxidase gene (choA) of a streptomycete was used as a model for studying heterologous gene expression in Streptococcus thermophilus, an essential bacterium in dairy food fermentations. The vectors pER82 and pER82P were developed from the 2.2-kb indigenous plasmid (pER8) of S. thermophilus ST108, and sP1, a 51-bp synthetic promoter patterned after a chromosomal sequence of S. thermophilus. The presence of sP1 promoter in pER82PbCOb with the choA insert aligned with the cat gene was essential for the intracellular production of cholesterol oxidase. The pER82PbCOb was apparently stable in S. thermophilus with no detectable evidence of deletion mutational events.

Construction and characterization of shuttle plasmids for lactic acid bacteria and Escherichia coli.

The chimeric plasmid pBN183 was first constructed in Escherichia coli by ligating the BamHI-digested E. coli plasmid pBR322 and a Bg/II-linearized streptococcal plasmid, pNZ18. The pBN183 transformed E. coli to ApR at a frequency of (8.2 +/- 1.2) x 10(5) colony forming units (CFU)/microgram DNA. Electrotransformation of Streptococcus thermophilus with pBN183 yielded CmR, ApS clones at a frequency of (2.6 +/- 0.3) x 10(1) CFU/microgram DNA. Plasmid screening with pBN183-transformed S. thermophilus clones revealed that ca. 70% of these transformants contained deleted plasmids. Plasmid pBN183A, a pBN183 deletion mutant lacking one copy of a tandemly arranged, highly homologous DNA sequence, was isolated for further study. It transformed E. coli to ApR and S. thermophilus to CmR with frequencies of (4.8 +/- 0.1) x 10(5) and (8.1 +/- 0.2) x 10(2) CFU/microgram DNA, respectively. Screening of S. thermophilus transformants did not show the presence of deleted plasmids. Based on the structure of pBN183A, a new shuttle plasmid, pDBN183, was constructed from pBN183 by removal of the small (1.2 kb) Sa/I fragment. Transformation frequencies of pDBN183 were (5.0 +/- 1.3) x 10(5) and (4.6 +/- 0.2) x 10(2) CFU/microgram DNA with E. coli and S. thermophilus, respectively. In contrast to the parent pBN183, only 17% of the pDBN183-transformed S. thermophilus contained deleted plasmids. Plasmid copy numbers of the three vectors in E. coli were estimated at 17-18 per chromosome. The three plasmids conferred ApR and CmR to E. coli, but only CmR to S. thermophilus. The insertion of a Streptomyces cholesterol oxidase gene (choA) into pDBN183 did not affect the plasmid's stability in Lactobacillus casei, but resulted in deletion of the recombinant DNA in S. thermophilus.

Transfer and expression of a Streptomyces cholesterol oxidase gene in Streptococcus thermophilus.

The recombinant plasmid pNCO937 (8.1 kbp) containing a Streptomyces sp. cholesterol oxidase gene was introduced into Streptococcus thermophilus by electrotransformation. Transformation frequency was 7.2 x 10(5) colony forming units/micrograms of DNA. The presence of the cholesterol oxidase gene in S. thermophilus was confirmed with Southern blot analysis using a biotinylated probe. Thin-layer chromatographic analysis showed the expression of the Streptomyces cholesterol oxidase gene resulting in the oxidation of cholesterol to 4-cholesten-3-one. S. thermophilus may be a suitable host for the expression of other genes regulating prokaryotic cholesterol metabolism.

Genetic transformation of Streptococcus thermophilus by electroporation.

A rapid and convenient electroporation procedure was developed for the genetic transformation of intact cells of Streptococcus thermophilus with various species of plasmid DNA. Transformation frequency was influenced by the capacitance and voltage selected for electric pulsing, the pH and composition of the electroporation medium and the molecular mass of the transforming DNA. Electroporation is a simple and effective technique to introduce plasmid DNA into S. thermophilus and useful in the development of recombinant DNA technology for this important industrial microorganism.

Adaptability of Streptococcus thermophilus to Lactose, Glucose and Galactose.

Significant differences in growth response to lactose, glucose and galactose were found among different strains of Streptococcus thermophilus . Some strains fermented all three carbohydrates (group A), whereas other strains utilized lactose and glucose only (group B) and one strain grew on lactose alone (group C). Characteristically, most lactose-grown strains in either group A or group B showed slow adaptation to either glucose or galactose after transfer to a medium containing either of these carbohydrates. Growth on glucose did not influence growth patterns following the transfer of either group A or group B strains to lactose broth. Lactose-grown group A and group B strains were restricted in growth following addition of galactose to the medium, whereas glucose-grown strains were not. The results suggested differences in carbohydrate transport mechanisms and utilization.

Thermal inactivation of staphylococcal enterotoxins B and C.

Thermal inactivation profiles of staphylococcal enterotoxins B (SEB) and C (SEC) at 80, 100, and 121 C showed that SEC is more resistant than SEB to heat. After 24 h of incubation at 25 C, some reactivation (recovery of serological reactivity) occurred in toxins that had been inactivated by heat. If the toxin was stirred during heating, reactivation did not occur. An examination of the reactivation kinetics of heat-treated SEC showed that reactivation was temperature dependent. At 25 C, the incubation temperature of heat-treated crude SEC (80 C for 10 min), 100% reactivation occurred after 24 h, whereas at 4 C only slight reactivation was observed. We and others observed that heat-treated toxins initially lost more serological activity when heated at a low temperature (80 C) than at a higher temperature (100 C); in the present study we demonstrate that this is a reversible phenomenon.