Characterization of prophages containing "evolved" Dit/Tal modules in the genome of Lactobacillus casei BL23.

Lactic acid bacteria (LAB) have many applications in food and industrial fermentations. Prophage induction and generation of new virulent phages is a risk for the dairy industry. We identified three complete prophages (PLE1, PLE2, and PLE3) in the genome of the well-studied probiotic strain Lactobacillus casei BL23. All of them have mosaic architectures with homologous sequences to Streptococcus, Lactococcus, Lactobacillus, and Listeria phages or strains. Using a combination of quantitative real-time PCR, genomics, and proteomics, we showed that PLE2 and PLE3 can be induced-but with different kinetics-in the presence of mitomycin C, although PLE1 remains as a prophage. A structural analysis of the distal tail (Dit) and tail associated lysin (Tal) baseplate proteins of these prophages and other L. casei/paracasei phages and prophages provides evidence that carbohydrate-binding modules (CBM) located within these "evolved" proteins may replace receptor binding proteins (RBPs) present in other well-studied LAB phages. The detailed study of prophage induction in this prototype strain in combination with characterization of the proteins involved in host recognition will facilitate the design of new strategies for avoiding phage propagation in the dairy industry.

Isolation and phenotypic characterization of Lactobacillus casei and Lactobacillus paracasei bacteriophage-resistant mutants.

AIMS: To isolate and characterize bacterial strains derived from Lactobacillus casei and Lactobacillus paracasei strains and resistant to phage MLC-A. METHODS AND RESULTS: Two of nine assayed strains rendered resistant mutants with recovery efficiencies of 83% (Lact. paracasei ATCC 27092) and 100% (Lact. casei ATCC 27139). DNA similarity coefficients (RAPD-PCR) confirmed that no significant genetic changes occurred while obtaining resistant mutants. Neither parent nor mutant strains spontaneously released phages. Phage-resistant mutants were tested against phages PL-1, J-1, A2 and MLC-A8. Lactobacillus casei ATCC 27092 mutants showed, overall, lower phage resistance than Lact. paracasei ATCC 27092 ones, but still higher than that of the parent strain. Lactobacillus paracasei ATCC 27092 mutants moderately adsorbed phage MLC-A only in calcium presence, although their parent strain successfully did it with or without calcium. Adsorption rates for Lact. casei ATCC 27139 and its mutants were highly influenced by calcium. Again, phage adsorption was higher on the original strain. CONCLUSIONS: Several isolates derived from two Lact. casei and Lact. paracasei strains showed resistance to phage MLC-A but also to other Lact. casei and Lact. paracasei phages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights isolation of spontaneous bacteriophage-resistant mutants from Lact. casei and Lact. paracasei as a good choice for use in industrial rotation schemes.

Widely distributed lysogeny in probiotic lactobacilli represents a potentially high risk for the fermentative dairy industry.

Prophages account for most of the genetic diversity among strains of a given bacterial species, and represent a latent source for the generation of virulent phages. In this work, a set of 30 commercial, collection and dairy-isolated Lactobacillus casei group strains were used. A species-specific PCR assay allowed a reclassification, mainly of strains previously considered Lactobacillus casei, into either Lactobacillus paracasei or Lactobacillus rhamnosus. All the strains were induced with mitomycin C, allowing direct recovering of phage DNA in 25 cases, which corroborates the widely occurrence of lysogeny on Lactobacillus genomes, including probiotic strains of Lactobacillus casei group. Ten out of 11 commercial strains studied contained prophages, evidencing the potential risks of their use at industrial scale. Strains were also induced by treatment with different concentrations of hydrogen peroxide but, however, this agent was not able to evidence a prophage release for any of the strains tested. According to a RAPD-PCR fingerprinting with M13, 1254 and G1 primers, most of the commercial strains presented a high degree of homology and, regarding BglII- and BamHI-restriction profiles of phage DNA, six of them harboured the same prophage. Surprisingly, both Lactobacillus paracasei ATCC 27092 and Lactobacillus paracasei ATCC 27139 shared a second prophage with both an INLAIN collection and a commercial Lactobacillus paracasei strains, whereas two collection strains shared a third one. On the other hand, mitomycin C-inducible prophages were detected only on about a half of the strains isolated from dairy products, which had (with only one exception) from moderate to high correlation coefficients according to RAPD-PCR fingerprinting. After induction, supernatants were filtered and tested against nine Lactobacillus strains of the set sensitive to previously assayed virulent phages, allowing isolation of two new virulent phages: ф iLp1308 and ф iLp84. Both phages were able to lyse all but one strains sensitive to previously assayed phage MLC-A.

PCR method for detection and identification of Lactobacillus casei/paracasei bacteriophages in dairy products.

Bacteriophage infections of starter lactic acid bacteria (LAB) pose a serious risk to the dairy industry. Nowadays, the expanding use of valuable Lactobacillus strains as probiotic starters determines an increase in the frequency of specific bacteriophage infections in dairy plants. This work describes a simple and rapid Polymerase Chain Reaction (PCR) method that detects and identifies bacteriophages infecting Lactobacillus casei/paracasei, the main bacterial species used as probiotic. Based on a highly conserved region of the NTP-binding genes belonging to the replication module of L. casei phages phiA2 and phiAT3 (the only two whose genomes are completely sequenced), a pair of primers was designed to generate a specific fragment. Furthermore, this PCR detection method proved to be a useful tool for monitoring and identifying L. casei/paracasei phages in industrial samples since specific PCR signals were obtained from phage contaminated milk (detection limit: 10(4) PFU/mL milk) and other commercial samples (fermented milks and cheese whey) that include L. casei/paracasei as probiotic starter (detection limit: 10(6) PFU/mL fermented milk). Since this method can detect the above phages in industrial samples and can be easily incorporated into dairy industry routines, it might be readily used to earmark contaminated milk for use in processes that do not involve susceptible starter organisms, or processes which involve phage-deactivating conditions.

Phages of Lactobacillus casei/paracasei: response to environmental factors and interaction with collection and commercial strains.

AIM: To investigate the influence of several environmental factors on the viability and cell-adsorption for two Lactobacillus casei/paracasei bacteriophages (PL-1 and J-1). METHODS AND RESULTS: Both phages showed a remarkably high specificity of species, sharing similar host spectra. Two phages and four sensitive strains were used to conform five phage/strain systems. Each showed a particular behaviour (burst size: ranging from 32 to 160 PFU/infective centre; burst time: 120-240 min and latent time: 5-90 min). For both phages, the viability was not significantly affected from pH 4 to 11 (room temperature) and from pH 5 to 10 (37 degrees C). Adsorption rates were not influenced by calcium ions, but decreased after the thermal inactivation of cells. Adsorption rates were high between 0 and 50 degrees C with maximum values at 30 degrees C and pH 6. System PL-1/Lact. paracasei A showed noticeable differences in comparison with the others, being times required to reach 90% of adsorption of 4 h and lower than 45 min, respectively. CONCLUSIONS: The data obtained in this work demonstrated that environmental parameters can influence the viability and cell adsorption rates of Lact. casei/paracasei phages. The extent of this influence was phage dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the enlargement of the currently scarce knowledge of phages of probiotic bacteria.