Sensitivity of capsular-producing Streptococcus thermophilus strains to bacteriophage adsorption.

AIMS: To determine whether the presence and type of exopolysaccharides (EPS), slime-EPS or capsular, and the structural characteristics of the polymers produced by Streptococcus thermophilus strains could interfere with or be involved in phage adsorption. METHODS AND RESULTS: Phage-host interactions between eight EPS-producing Strep. thermophilus strains (CRL419, 638, 804, 810, 815, 817, 821, 1190) and five streptococcus specific phages (phiYsca, phi3, phi5, phi6, phi8) isolated from Argentinean faulty fermentation failed yoghurts were evaluated. No relationship was found between the EPS chemical composition and the phage sensitivity/resistance phenotype. In general, the capsular-producing strains were more sensitive to phage attacks than the noncapsular-producing strains. Streptococcus thermophilus CRL1190 (capsular-producing) was the only strain sensitive to all bacteriophages and showed the highest efficiency of plating. Phage adsorption to a capsular-negative, EPS low-producing mutant of strain CRL1190 was reduced, especially for phiYcsa and phi8. CONCLUSIONS: The presence of capsular polysaccharide surrounding the cells of Strep. thermophilus strains could play a role in the adsorption of specific phages to the cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Capsular-producing Strep. thermophilus strains should be evaluated for their bacteriophage sensitivity if they are included in starter cultures for the fermented food industry.

Selection of bacteriocin producer strains of lactic acid bacteria from a dairy environment.

Two strains showing bacteriocin production were selected from a total of 206 lactic acid bacteria isolated from samples of milk, milk serum, whey and homemade cheeses in Southern Cordoba, Argentina. This property was detected by means of well diffusion assays. The strains were identified as Enterococcus hirae and Enterococcus durans. The protein nature of those substances was proved by showing their sensitivity to type IV and XXV proteases, papaine, trypsin, pepsin and K proteinase. The bacteriocins inhibited the growth of Listeria monocytogenes, Bacillus cereus, Clostridium perfringes and two strains of Staphylococcus aureus, an A-enterotoxin and a B-enterotoxin producers. All of these bacteria are common pathogens usually associated with food borne diseases (ETA). These lactic acid bacteria or their bacteriocins could be suitable candidates for food preservation and specially useful in the our regional dairy industry.

Lactic acid bacteria and their effect on the immune system.

Lactic acid bacteria (LAB) are present in the intestine of most animals. The beneficial role played by these microorganisms in the humans and other animals, including the effect on the immune system, has been extensively reported. They are present in many foods and are frequently used as probiotics to improve some biological functions in the host. The activation of the systemic and secretory immune response by LAB requires many complex interactions among the different constituents of the intestinal ecosystem (microflora, epithelial cells and immune cells). Through different mechanisms they send signals to activate immune cells. Thus the knowledge of the normal intestinal microflora, the contribution of LAB and their role in the numerous functions in the digestive tract as well as the functioning of the mucosal immune system form the basis for the study and selection of a probiotic strain with immunostimulatory properties. In the selection of LAB by their immunostimulatory capacity it helps to know not only the effect which they have on the mucosal immune system, but the specific use to which these oral vaccine vectors are being put. Although there are reports of the protection of animals and humans against diseases such as microbial infections and cancer, more work remains to be done on the factors affecting the design of oral vaccine vectors and the use of LAB for therapeutic purposes. The basic knowledge of LAB immunostimulation and the criteria for selection of LAB by their immunostimulatory capacity, will be extensively discussed and appraised in this review.

Isolation and characterization of a slowly milk-coagulating variant of Lactobacillus helveticus deficient in purine biosynthesis.

A slowly milk-coagulating variant (Fmc(-)) of Lactobacillus helveticus CRL 1062, designated S1, was isolated and characterized. Strain S1 possessed all the known essential components required to utilize casein as a nitrogen source, which include functional proteinase and peptidase activities as well as functional amino acid, di- and tripeptide, and oligopeptide transport systems. The amino acid requirements of strain S1 were similar to those of the parental strain. However, on a purine-free, chemically defined medium, the growth rate of the Fmc(-) strain was threefold lower than that of the wild-type strain. L. helveticus S1 was found to be defective in IMP dehydrogenase activity and therefore was deficient in the ability to synthesize XMP and GMP. This conclusion was further supported by the observation that the addition of guanine or xanthine to milk, a substrate poor in purine compounds, restored the Fmc(+) phenotype of L. helveticus S1.

Nutritional requirements and nitrogen-dependent regulation of proteinase activity of Lactobacillus helveticus CRL 1062.

The nutritional requirements of Lactobacillus helveticus CRL 1062 were determined with a simplified chemically defined medium (SCDM) and compared with those of L. helveticus CRL 974 (ATCC 15009). Both strains were found to be prototrophic for alanine, glycine, asparagine, glutamine, and cysteine. In addition, CRL 1062 also showed prototrophy for lysine and serine. The microorganisms also required riboflavin, calcium pantothenate, pyridoxal, nicotinic acid, and uracil for growth in liquid SCDM. The growth rate and the synthesis of their cell membrane-bound serine proteinases, but not of their intracellular leucyl-aminopeptidases, were influenced by the peptide content of the medium. The highest proteinase levels were found during cell growth in basal SCDM, while the synthesis of this enzyme was inhibited in SCDM supplemented with Casitone, Casamino Acids, or beta-casein. Low-molecular-mass peptides (<3,000 Da), extracted from Casitone, and the dipeptide leucylproline (final concentration, 5 mM) play important roles in the medium-dependent regulation of proteinase activity. The addition of the dipeptide leucylproline (5 mM) to SCDM reduced proteinase activity by 25%.

Characterization of natural isolates of Lactobacillus strains to be used as starter cultures in dairy fermentation.

The technological relevant characteristics of five homofermentative lactobacilli strains, isolated from natural fermented hard cheeses, were studied. Isolates CRL 581 and CRL 654, from Argentinian artesanal hard cheeses, and isolates CRL 1177, CRL 1178, and CRL 1179, from Italian Grana cheeses, were identified as Lactobacillus delbrueckii subsp. lactis and Lactobacillus helveticus, respectively, by physiological and biochemical tests, SDS-PAGE of whole-cell proteins and sequencing of the variable (V1) region of the 16S ribosomal DNA. All strains showed high levels of beta-galactosidase activity. However, proteolytic activity varied widely among isolates. Strains CRL 581, CRL 654, and CRL 1177 hydrolyzed alpha- and beta-caseins and were able to coagulate reconstituted skim milk in less than 16 h at 42 degrees C. According to the substrate specificity, these proteinases have a caseinolytic activity comparable to that of the P(III)-type of lactococcal proteinases. No strains produced inhibitor substances (bacteriocin) and all were insensitive to attack by 14 L. helveticus- and L. delbrueckii subsp. lactis-specific bacteriophages.

Deoxyribonuclease activities in Lactobacillus delbrueckii.

DNase activity was examined in the extracellular and subcellular fractions of six non-transformable strains belonging to Lactobacillus delbrueckii subsp. lactis (L. lactis) and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and compared with the activity present in Lactobacillus johnsonii NCK 65, a transformable strain of Lactobacillus. In the extracellular fraction of the L. delbrueckii strains, a common protein band of 36 kDa was detected, while a band of 29 kDa was found in the same fraction of L. johnsonii. No nuclease activity was detected in the cytoplasmic fraction of this strain, indicating that the localization of the DNase activity could be a key factor in the uptake of foreign DNA.

Identification and nucleotide sequence of genes involved in the synthesis of lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL 705.

The structural gene determinants of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705, have been amplified from a plasmid of approximately 35 kb and sequenced. Lactocin 705 is a class IIb bacteriocin, whose activity depends upon the complementation of two peptides (705alpha and 705beta) of 33 amino acid residues each. These peptides are synthesized as precursors with signal sequences of the double-glycine type, which exhibited high identities with the leader peptides of plantaricin S and J from Lactobacillus plantarum, brochocin C from Brochotrix campestris, sakacin P from Lactobacillus sake, and the competence stimulating peptides from Streptococcus gordonii and Streptococcus mitis. However, the two mature bacteriocins 705alpha and 705beta do not show significant similarity to other sequences in the databases.

Physical mapping and partial genetic characterization of the Lactobacillus delbrueckii subsp. bulgaricus bacteriophage lb539.

A restriction map was constructed of the 37 kb genome of the temperate Lactobacillus delbrueckii subsp. bulgaricus bacteriophage lb539. Restriction analysis and Southern hybridization experiments detected variable levels of homologous regions among the genomes of lb539 and the L. delbrueckii reference phages LL-H (virulent) and mv4 (temperate). The principal homology was observed at the regions encoding the structural proteins. These studies allowed us to construct a partial genetic map of phage lb539 for lysin, the main structural tail protein and the packaging region genes. Furthermore, a short 1.5 kb DNA fragment of the prolate-headed JCL1032 phage genome was observed to be highly homologous with the DNA of the isometric-headed lb539, mv4 and LL-H phages. The described distribution of the homologous regions between the genomes of the phages lb539, LL-H, mv4 and JCL1032 presented here supports the modular evolution theory of the bacteriophages.

Venezuelan equine encephalomyelitis and African horse sickness. Current status and review.

The arthropod-borne virus (arbovirus) diseases of livestock have worldwide impact. The prevention of an introduction of an exotic disease and the control of one subsequent to an introduction will require the attention, cooperation, and support of the livestock industry, regulatory agencies, and researchers. The most effective protection of our livestock industries is to prevent the introduction of an exotic disease agent. This implies complete restriction of animal imports and exports. However, "zero risk" is an unacceptable option in today's world of internationally integrated and interdependent agriculture. Scientifically sound and factually based regulatory decisions must be combined with continued vigilance and preparedness, as well as appropriately directed surveillance and research, to protect the world agricultural marketplace. Two exotic arbovirus diseases that are of current concern to the livestock industries and regulatory officials of the North American and Caribbean Basin countries are VEE and AHS. Devastating epizootics of VEE in equines have occurred frequently in the Western Hemisphere, but no recent epizootic activity has been documented. Naturally occurring foci of sylvatic, equine nonpathogenic VEE virus subtypes, however, do exist in the tropical countries of the hemisphere. The relationship of these sylvatic virus foci to the origin of equine virulent epizootic VEE virus subtypes is unknown. AHS epizootics had been confined to Africa, the Middle East, and the Indian subcontinent until recent outbreaks in 1966 and 1987-1990 in Spain. With the recurrence of AHS in Spain for the past four years, concern about the possible introduction into potential vector species and equines in the Western Hemisphere has increased. This review addresses the current VEE and AHS virus activity and the potential for outbreaks in the Western Hemisphere.