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PubMed2005

Detection and characterization of Streptococcus thermophilus bacteriophages by use of the antireceptor gene sequence.

In the dairy industry, the characterization of Streptococcus thermophilus phage types is very important for the selection and use of efficient starter cultures. The aim of this study was to develop a characterization system useful in phage control programs in dairy plants. A comparative study of phages of different origins was initially performed based on their morphology, DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and lifestyles and on the presence of a highly conserved DNA fragment of the replication module. However, these traditional criteria were of limited industrial value, mainly because there appeared to be no correlation between these variables and host ranges. We therefore developed a PCR method to amplify VR2, a variable region of the antireceptor gene, which allowed rapid detection of S. thermophilus phages and classification of these phages. This method has a significant advantage over other grouping criteria since our results suggest that there is a correlation between typing profiles and host ranges. This association could be valuable for the dairy industry by allowing a rational starter rotation system to be established and by helping in the selection of more suitable starter culture resistance mechanisms. The method described here is also a useful tool for phage detection, since specific PCR amplification was possible when phage-contaminated milk was used as a template (detection limit, 10(5) PFU ml(-1)).

Animals↗
PubMed2005

Characterization of the cro-ori region of the Streptococcus thermophilus virulent bacteriophage DT1.

The virulent cos-type Streptococcus thermophilus phage DT1 was previously isolated from a mozzarella whey sample, and its complete genomic sequence is available. The putative ori of phage DT1 is characterized by three inverted and two direct repeats located in a noncoding region between orf36 and orf37. As the replication ability of the putative ori and flanking genes could not be established, its ability to confer phage resistance was tested. When ori is cloned on a high-copy-number plasmid, it provides protection to S. thermophilus strains against phage infection during milk fermentation. This protection is phage specific and strain dependent. Then, a detailed transcriptional map was established for the region located between the cro-like gene (orf29) and the ori. The results of the Northern blots indicated that the transcription of this region started 5 min after the onset of phage infection. Comparative analysis of the expression of the cro-ori region in the three S. thermophilus cos-type phages DT1, Sfi19 (virulent), and Sfi21 (temperate) reveals significant differences in the number and size of transcripts. The promoter upstream of orf29 was further investigated by primer extension analysis, and its activity was confirmed by a chloramphenicol acetyltransferase assay, which showed that the phage promoter is more efficient than the constitutive bacterial promoter of the S. thermophilus operon encoding the general proteins of the phosphoenolpyruvate:sugar phosphotransferase system. However, the phage promoter is less efficient than the pts promoter in Lactococcus lactis and in Escherichia coli.

Animals↗
PubMed2003

Phage lytic enzyme Cpl-1 as a novel antimicrobial for pneumococcal bacteremia.

Streptococcus pneumoniae is becoming increasingly antibiotic resistant worldwide, and thus new antimicrobials are badly needed. We report the use of Cpl-1, the lytic enzyme of a pneumococcal bacteriophage, as an intravenous therapy for pneumococcal bacteremia in a mouse model. A 2000- microg dose of Cpl-1 reduced pneumococcal titers from a median of log(10) 4.70 CFU/ml to undetectable levels (<log(10) 2.00 CFU/ml) within 15 min. This dose given 1 h after intravenous infection led to 100% survival at 48 h, compared to the 20% survival of buffer-treated controls. In advanced bacteremia, treatment with two doses at 5 and 10 h still resulted in significantly longer survival (P < 0.0001) and a hazard ratio of 0.29 (95% confidence interval, 0.04 to 0.35). The enzyme is immunogenic, but the treatment efficacy was not significantly diminished after previous intravenous exposure of mice and hyperimmune rabbit serum did not neutralize the activity. Cpl-1 is also very effective as a topical nasal treatment against colonization by S. pneumoniae. In vitro, the enzyme is active against many serotypes of S. pneumoniae, independent of their penicillin resistance, and it is very specific for this species. Bacteriophage enzymes are unusual but extremely effective antimicrobials and represent a new weapon against infections with resistant bacteria.

Animals↗
PubMed1975

A large new Streptococcus bacteriophage.

Phage VD13 possesses a large, elongated head and a long, non-contractile tail. It is active on group D streptococci and contains double-stranded DNA. The phage produces several rare kinds of head malformations, notably polymorphic mottled structures and giant heads which probably contain DNA.

Bacteriophages↗
PubMed2003

Selection of bacteriophage-resistant mutants of Streptococcus thermophilus.

Phage-resistant mutants have been isolated from Streptococcus thermophilus. Selection was carried out using anti-phage antibodies or Hoechst 33258-labelled phages. Two mutants out of eight tested displayed reduced acidifying capacity. Selection of the bacteria that extruded more rapidly the fluorochrome 5-6-carboxyfluorescein diacetate (CFDA) restored the acidifying capacity of these two mutants to the level of the parental strains. Mutants displaying phage resistance and good acidifying capacity were obtained in 4-5 days. New phages that are able to overcome the protection mechanisms of the existing bacteria arise continually in the dairy environment. The procedures described here permit to replace promptly the starter culture susceptible to newly emerged phages with a resistant one.

Adsorption↗
PubMed1987

DNA-free liposome-assisted transfection of Streptococcus faecium SF 77R protoplasts.

Two virulent phages of Streptococcus faecium SF 77R were used in DNA-free liposome assisted polyethylene-glycol-mediated protoplast transfection. This procedure, established to develop an effective gene-cloning system, optimized the conditions for DNA uptake by the protoplasts of Str. faecium. Transfection frequencies of up to 10(7)-10(8) P.F.U./microgram of DNA were achieved.

Bacteriophages↗
PubMed1996

Cloning, sequence analysis, and expression of the genes encoding lytic functions of Bacteriophage phi g1e.

The lysis genes of a Lactobacillus phage phi g1e were cloned, sequenced, and expressed in Escherichia coli. Nucleotide sequencing of a 3813-bp phi g1e DNA revealed five successive open reading frames (ORF), Rorf50, Rorf118, hol, and lys and Rorf175, in the same DNA strand. By comparative analysis of the DNA sequence, the putative hol product (holin) has an estimated molecular weight is 14.2 kDa, and contains two potential transmembrane helices and highly charged N- and C-termini, resembling predicted holins (which are thought to be a cytoplasmic membrane-disrupting protein) encoded by other phages such as mv1 from Lactobacillus bulgaricus, phi adh from Lactobacillus gasseri, as well as monocins from Listeria. On the other hand, the putative phi g1e lys product (lysin) of 48.4 kDa shows significant similarity with presumed muramidase, known as a cell wall peptidoglycandegrading enzyme, encoded by the Lactobacillus phage mv1 and phi adh, the Lactococcus lactis phage phi LC3, and the Streptococcus pneumoniae phages Cp-1, Cp-7 and Cp-9. When expressed in E. coli, the phi g1e lysin and/or holin decreased the cell turbidity significantly, suggesting that the phi g1e hol-lys system is involved in cytolytic process.

Amino Acid Sequence↗
PubMed1995

Expression of a Lactococcus lactis Phage Resistance Mechanism by Streptococcus thermophilus.

The 7.8-kb lactococcal plasmid pSRQ700 encodes the LlaII restriction/modification system which recognizes and cleaves the sequence 3(prm1)-GATC-5(prm1). When the plasmid pSRQ700 is introduced into a phage-sensitive Lactococcus lactis strain, strong phage resistance is conferred by the LlaII system. In this report, we show that pSRQ700 cannot replicate in Streptococcus thermophilus. However, if cloned into the vector pNZ123, the native LlaII system is expressed and strong phage resistance is conferred to various industrial S. thermophilus strains. Resistance against phages isolated from yogurt and mozzarella wheys was observed. To our knowledge, this is the first report of increased phage resistance in S. thermophilus.

Journal Article↗
PubMed2000

Selection of recombinant antibodies specific for pathogenic Streptococcus suis by subtractive phage display.

A semisynthetic antibody phage display library was used to select recombinant antibodies directed against surface components of a pathogenic strain of Streptococcus suis serotype 2 and against extracellular factor (EF), a protein known to be exclusively associated with pathogenic S. suis serotype 2 strains. Three distinct monoclonal phage antibodies directed against conformational epitopes of surface protein components of S. suis were selected. In addition, three different monoclonal phage antibodies were isolated that recognized EF. To isolate antibody fragments that recognize epitopes specific for a pathogenic S. suis serotype 2 strain, compared to a nonpathogenic serotype 2 strain, we applied a subtractive selection procedure. With this procedure, only one distinct phage antibody was found, and it was shown to be directed against EF. This demonstrates the selectivity of the applied procedure and confirms that EF is indeed differentially expressed by pathogenic and nonpathogenic strains. It also shows that EF is a very dominant antigen in phage antibody selections.

Amino Acid Sequence↗
PubMed1974

Morphology of the bacteriophages of lactic streptococci.

Electron microscope studies have been made of a number of phages of lactic streptococci, seven of which were phages of Streptococcus lactis C10. Two of the phages are thought to be identical; five have been classified by the method of Tikhonenko as belonging to group IV (phages with noncontractile tails) with type III tail plates; one belongs to group V (phages with tails possessing a contractile sheath). Both prolate polyhedral heads and isometric polyhedral heads are represented among the group IV phages. The phage drc3 of S. diacetilactis DRC3 has been shown to have similar structure to the group IV phages of S. lactis C10 with prolate polyhedral heads. The phages ml1, hp, c11, and z8 of the S. cremoris strains ML1, HP, C11, and Z8, respectively, were shown to belong to the group IV phages with type III tail plates by the method of Tikhonenko. All had octahedral heads and tended to be larger than most of the other phages studied.

Bacteriophages↗
PubMed1985

Partial purification and some properties of phi C2(W) lysin, a lytic enzyme produced by phage-infected cells of Streptococcus lactis C2.

The lytic enzyme present in phi C2(W) lysates was isolated by means of ion-exchange chromatography and further purified by gel filtration and ultrafiltration. The phage enzyme had an apparent pH optimum of 6.5-6.9 and was rapidly inactivated at temperatures greater than 47 degrees C. The apparent temperature optimum was 37 degrees C and Q10 and Ea values over the range 22-32 degrees C were 2.5 and 69.2 kJ/mol respectively. Monovalent and divalent cations activated the enzyme. Reduced -SH groups on the enzyme were required for lytic activity. Gel filtration revealed a mol. wt of approximately 46000. Strain-dependent differences in sensitivity of group N lactic streptococci to lysin were found. Group D streptococci were also lysed. Strains of three species of Leuconostoc, two species of Lactobacillus, one strain of Escherichia coli and of Micrococcus lysodeikticus were apparently resistant. Analysis of cell wall degradation products gave results which were consistent with the lysin having the specificity of an N-acetylmuramidase.

Bacteriophages↗
PubMed1987

Molecular characterization and comparison of 38 virulent and temperate bacteriophages of Streptococcus lactis.

Thirty-three virulent and five temperate phages of Streptococcus lactis and Streptococcus cremoris were differentiated into three groups by DNA homology. A complete lack of DNA homology was demonstrated between the phage groups. Within each group, large parts of the phage genomes were homologous except for a few phages. One group consisted of five temperate and two virulent phages suggesting that virulent phages isolated during abnormal fermentations and temperate phages isolated after induction from lactic streptococcal starter cultures may be related to one another. We observed a good correlation between the grouping of phages by DNA homology and by their protein composition since within the same DNA homology group, the protein composition of a phage exhibited some similarities with that of the other phages of the group. Therefore, the DNA homologies seemed to be located, at least, in the region coding for the structural proteins. By immunoblotting, we confirmed the relatedness between the proteins of the phages belonging to the same DNA homology group. The important host range factor in bacterium-phage interactions appeared to be an unreliable criterion in determining phage taxonomy.

Bacteriophages↗