Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei.

Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: CL1, CL2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (CL1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages CL1, CL2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages CL1, CL2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts.

Characterization of two temperate Lactobacillus paracasei bacteriophages: morphology, kinetics and adsorption.

BACKGROUND/AIMS: Adsorption and kinetic parameters, latent period, burst size and burst time, are characteristics of phage/host systems and can be affected by several environmental factors. As only few studies have focused on temperate dairy phages, we characterized these parameters on temperate Lactobacillus paracasei phages Φ iLp84 and Φ iLp1308, infective for probiotic strains. METHODS: Phages were characterized by transmission electron microscopy and genomic DNA restriction. Adsorption under different environmental conditions, phage kinetics and efficiency of plating (EOP) were determined using the double-layer titration method. RESULTS: Phages Φ iLp84 and Φ iLp1308 belong to the Siphoviridae family and have genome sizes of 38 and 34 kbp, respectively. Adsorption was affected by calcium concentration, pH, temperature and host viability, and reached a limit at very high multiplicity of infection. Latency, burst time and burst size were of 85 min, 131 min and 46 for Φ iLp84, and 51 min, 92 min and 28 for Φ iLp1308, respectively, at 37°C. A clear influence of temperature on phage kinetics was observed. Regarding EOP, Φ iLp84 produced plaques on only 1 out of 8 strains tested. CONCLUSION: Noticeable differences in adsorption, kinetics and EOP were found for two morphologically identical temperate L. paracasei phages of similar origin.

A fast PCR-based method for the characterization of prophage profiles in strains of the Lactobacillus casei group.

Lysogeny is widespread among Lactobacillus strains of the casei group (L. casei, L. paracasei and L. rhamnosus), and prophages account for most strain-specific DNA. Numerous PCR based methods have been developed to detect free phages of lactic acid bacteria, but they do not take in consideration prophages. In this study, a new PCR method for the detection of lysogeny was developed using genome sequences of L. casei group strains (including BL23) and bacteriophages. Nine pairs of primers were designed to selectively amplify the highly conserved prophage iA2 (pairs #1-#3) and fragments of two groups phages of temperate origin: CL1/CL2/iLp1308/iLp84 (pairs #4 and #5) and Lrm1/J-1/PL-1/A2/AT3/Lc-Nu (pairs #6 to #9). Forty-nine strains of the casei group were subjected to PCR. Strains containing remnants of lytic phages outnumbered those containing iA2-related prophages. The combination of pair #2, annealing on the terminase large subunit (TLS), and pair #3, annealing on the helicase (forward) and a non-coding region (reverse), showed the best diagnostic performance for iA2-like prophages. For the assessment of remnants of phages CL1/CL2/iLp1308/iLp84, pair #4 (annealing on the TLS) was preferred over pair #5 (portal protein). Detection of phages Lrm1/J-1/PL-1/A2/AT3/Lc-Nu was optimal with primers of pair #6, designed on non-coding regions of phage genomes; pair #6 also evidenced a high conservation of certain prophage remnants. Overall, our PCR-based method successfully detected and discriminated groups of prophages or remnants in L. casei group strains.

Phages of dairy Leuconostoc mesenteroides: genomics and factors influencing their adsorption.

Phages infecting Leuconostoc mesenteroides strains can be overlooked during milk fermentation because they do not slowdown the acidification process. However, they can negatively impact the flavor profile of the final product. Yet, the information about these phages is still scarce. In this work, we investigated diverse factors influencing the adsorption of seven virulent Ln. mesenteroides phages, isolated from blue cheese manufacture in Argentina, to their host cells. The addition of calcium ions was generally necessary to observe complete cell lysis and plaque formation for four of the seven phages, but adsorption was very high even in the absence of this cation for all phages. The temperature barely influenced the adsorption process as it was high within the temperature range tested (0 to 50 °C). Moreover, the kinetics of adsorption were similar on viable and non-viable cells, revealing that phage adsorption does not depend on physiological state of the bacterial cells. The adsorption rates were also high at pH values from 4 to 9 for all Ln. mesenteroides phages. We also analyzed the complete genome sequences of two of these phages. Complete nucleotide analysis of phages Ln-8 and Ln-9 showed dsDNA genomes with sizes of 28.5 and 28.9 kb, and the presence of 45 and 48 open reading frames (ORFs), respectively. These genomes were highly similar to those of previously characterized Φ1-A4 (USA, sauerkraut, fermentation) and ΦLN25 (England, whey), both virulent Ln. mesenteroides phages. A detailed understanding of these phages will lead to better control strategies.

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.

Review: efficiency of physical and chemical treatments on the inactivation of dairy bacteriophages.

Bacteriophages can cause great economic losses due to fermentation failure in dairy plants. Hence, physical and chemical treatments of raw material and/or equipment are mandatory to maintain phage levels as low as possible. Regarding thermal treatments used to kill pathogenic bacteria or achieve longer shelf-life of dairy products, neither low temperature long time nor high temperature short time pasteurization were able to inactivate most lactic acid bacteria (LAB) phages. Even though most phages did not survive 90°C for 2 min, there were some that resisted 90°C for more than 15 min (conditions suggested by the International Dairy Federation, for complete phage destruction). Among biocides tested, ethanol showed variable effectiveness in phage inactivation, since only phages infecting dairy cocci and Lactobacillus helveticus were reasonably inactivated by this alcohol, whereas isopropanol was in all cases highly ineffective. In turn, peracetic acid has consistently proved to be very fast and efficient to inactivate dairy phages, whereas efficiency of sodium hypochlorite was variable, even among different phages infecting the same LAB species. Both alkaline chloride foam and ethoxylated non-ylphenol with phosphoric acid were remarkably efficient, trait probably related to their highly alkaline or acidic pH values in solution, respectively. Photocatalysis using UV light and TiO(2) has been recently reported as a feasible option to industrially inactivate phages infecting diverse LAB species. Processes involving high pressure were barely used for phage inactivation, but until now most studied phages revealed high resistance to these treatments. To conclude, and given the great phage diversity found on dairies, it is always advisable to combine different anti-phage treatments (biocides, heat, high pressure, photocatalysis), rather than using them separately at extreme conditions.