Evaluation of a novel cocktail of six lytic bacteriophages against Shiga toxin-producing Escherichia coli in broth, milk and meat.

Phages are potentially useful as antimicrobial agents in food, especially cocktails of different phages which may prevent the development of bacterial resistance. Biocontrol assays with a six-phage cocktail, which is lytic against DH5α, an enteropathogenic (EPEC) and two Shiga-toxigenic (STEC) Escherichia coli strains, were performed in Hershey-Mg broth, milk and meat at refrigerated (4 °C), room (24 °C) and abusive (37 °C) temperatures. At 4 °C, cell counts were significantly lower (2.2-2.8 log10 CFU/mL) when E. coli strains (∼109 CFU/mL) were challenged against the phage cocktail (∼109 PFU/mL) in Hershey-Mg broth after 24 h. However, reductions were higher (3.2-3.4 log10 CFU/mL) after a 48 h exposure for all the strains tested. In addition, reduction values reached up to 3.4 log10 CFU/mL (24 °C) and 3.6 log10 CFU/mL (37 °C) in challenge tests after 24 h, though the reductions achieved were slightly lower after 48 h for the four E. coli strains tested. In milk, the cocktail was highly effective since bacterial counts were below the detection limit (<101 CFU/mL) at 4 °C, while the reductions ranged from 2 to 4 log10 CFU/mL at 24 °C after a 24 h exposure. At 37 °C, DH5α was eliminated within 2 h, and an average cell decrease of 4 log10 CFU/mL was observed for the three pathogenic strains tested. When the assays were performed in meat, biocontrol values ranged from 0.5 to 1.0 log10 CFU/mL after 48 h at 4 °C, while a higher cell inactivation was achieved at 24 °C (2.6-4.0 log10 CFU/mL) and 37 °C (3.0-3.8 log10 CFU/mL). Furthermore, higher inactivation values for O157:H7 STEC (1.55 ±â€¯0.35 log10 CFU/mL) at 4 °C were obtained in meat when incubation was extended up to 6 days. As a conclusion, our six-phage cocktail was highly effective at 24 °C and 37 °C, though less effective at 4 °C in both food matrices evaluated. Thus, it might be applied against pathogenic EPEC and STEC strains to prevent foodborne diseases especially when the cold chain is lost.

Response of Leuconostoc strains against technological stress factors: Growth performance and volatile profiles.

The ability of twelve strains belonging to three Leuconostoc species (Leuconostoc mesenteroides, Leuconostoc lactis and Leuconostoc pseudomesenteroides) to grow under diverse sub-lethal technological stress conditions (cold, acidic, alkaline and osmotic) was evaluated in MRS broth. Two strains, Leuconostoc lactis Ln N6 and Leuconostoc mesenteroides Ln MB7, were selected based on their growth under sub-lethal conditions, and volatile profiles in RSM (reconstituted skim milk) at optimal and under stress conditions were analyzed. Growth rates under sub-lethal conditions were strain- and not species-dependent. Volatilomes obtained from the two strains studied were rather diverse. Particularly, Ln N6 (Ln. lactis) produced more ethanol and acetic acid than Ln MB7 (Ln. mesenteroides) and higher amounts and diversity of the rest of volatile compounds as well, at all times of incubation. For the two strains studied, most of stress conditions applied diminished the amounts of ethanol and acetic acid produced and the diversity and levels of the rest of volatile compounds. These results were consequence of the different capacity of the strains to grow under each stress condition tested.

Leuconostoc strains isolated from dairy products: Response against food stress conditions.

A systematic study about the intrinsic resistance of 29 strains (26 autochthonous and 3 commercial ones), belonging to Leuconostoc genus, against diverse stress factors (thermal, acidic, alkaline, osmotic and oxidative) commonly present at industrial or conservation processes were evaluated. Exhaustive result processing was made by applying one-way ANOVA, Student's test (t), multivariate analysis by Principal Component Analysis (PCA) and Matrix Hierarchical Cluster Analysis. In addition, heat adaptation on 4 strains carefully selected based on previous data analysis was assayed. The strains revealed wide diversity of resistance to stress factors and, in general, a clear relationship between resistance and Leuconostoc species was established. In this sense, the highest resistance was shown by Leuconostoc lactis followed by Leuconostoc mesenteroides strains, while Leuconostoc pseudomesenteroides and Leuconostoc citreum strains revealed the lowest resistance to the stress factors applied. Heat adaptation improved thermal cell survival and resulted in a cross-resistance against the acidic factor. However, all adapted cells showed diminished their oxidative resistance. According to our knowledge, this is the first study regarding response of Leuconostoc strains against technological stress factors and could establish the basis for the selection of "more robust" strains and propose the possibility of improving their performance during industrial processes.

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.

Phage adsorption and lytic propagation in Lactobacillus plantarum: could host cell starvation affect them?

BACKGROUND: Bacteriophages constitute a great threat to the activity of lactic acid bacteria used in industrial processes. Several factors can influence the infection cycle of bacteriophages. That is the case of the physiological state of host cells, which could produce inhibition or delay of the phage infection process. In the present work, the influence of Lactobacillus plantarum host cell starvation on phage B1 adsorption and propagation was investigated. RESULT: First, cell growth kinetics of L. plantarum ATCC 8014 were determined in MRS, limiting carbon (S-N), limiting nitrogen (S-C) and limiting carbon/nitrogen (S) broth. L. plantarum ATCC 8014 strain showed reduced growth rate under starvation conditions in comparison to the one obtained in MRS broth. Adsorption efficiencies of > 99 % were observed on the starved L. plantarum ATCC 8014 cells. Finally, the influence of cell starvation conditions in phage propagation was investigated through one-step growth curves. In this regard, production of phage progeny was studied when phage infection began before or after cell starvation. When bacterial cells were starved after phage infection, phage B1 was able to propagate in L. plantarum ATCC 8014 strain in a medium devoid of carbon source (S-N) but not when nitrogen (S-C broth) or nitrogen/carbon (S broth) sources were removed. However, addition of nitrogen and carbon/nitrogen compounds to starved infected cells caused the restoration of phage production. When bacterial cells were starved before phage infection, phage B1 propagated in either nitrogen or nitrogen/carbon starved cells only when the favorable conditions of culture (MRS) were used as a propagation medium. Regarding carbon starved cells, phage propagation in either MRS or S-N broth was evidenced. CONCLUSIONS: These results demonstrated that phage B1 could propagate in host cells even in unfavorable culture conditions, becoming a hazardous source of phages that could disseminate to industrial environments.

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.

Resistance of functional Lactobacillus plantarum strains against food stress conditions.

The survival of three Lactobacillus plantarum strains (Lp 790, Lp 813 and Lp 998) with functional properties was studied taking into account their resistance to thermal, osmotic and oxidative stress factors. Stress treatments applied were: 52 °C-15 min (Phosphate Buffer pH 7, thermal shock), H2O2 0.1% (p/v) - 30 min (oxidative shock) and NaCl aqueous solution at 17, 25 and 30% (p/v) (room temperature - 1 h, osmotic shock). The osmotic stress was also evaluated on cell growth in MRS broth added of 2, 4, 6, 8 and 10% (p/v) of NaCl, during 20 h at 30 °C. The cell thermal adaptation was performed in MRS broth, selecting 45 °C for 30 min as final conditions for all strains. Two strains (Lp 813 and Lp 998) showed, in general, similar behaviour against the three stress factors, being clearly more resistant than Lp 790. An evident difference in growth kinetics in presence of NaCl was observed between Lp 998 and Lp 813, Lp998 showing a higher optical density (OD570nm) than Lp 813 at the end of the assay. Selected thermal adaptation improved by 2 log orders the thermal resistance of both strains, but cell growth in presence of NaCl was enhanced only in Lp 813. Oxidative resistance was not affected with this thermal pre-treatment. These results demonstrate the relevance of cell technological resistance when selecting presumptive "probiotic" cultures, since different stress factors might considerably affect viability or/and performance of the strains. The incidence of stress conditions on functional properties of the strains used in this work are currently under research in our group.

Leuconostoc citreum MB1 as biocontrol agent of Listeria monocytogenes in milk.

Cell-free supernatant from Leuconostoc citreum MB1 revealed specific antilisterial activity. Preliminary studies demonstrated the proteinaceous, heat-stable, bacteriocin-like trait of the antimicrobial components present in the supernatant. Determination of the genes encoding bacteriocins by PCR and DNA sequencing led to amplification products highly homologous with leucocin A (found in diverse Leuconostoc species) and UviB (found in Leuc. citreum KM20) sequences. Additionally, antimicrobial activity of cell-free supernatant from Leuc. citreum MB1 was revealed by an inhibition halo of the SDS-PAGE gel subjected to a direct detection using Listeria monocytogenes as indicator strain. Different assays were carried out to assess the capacity of Leuc.citreum MB1 to control List. monocytogenes growth: (i) inactivation kinetics of the pathogen by antilisterial compounds present in concentrated cell-free supernatant from Leuc. citreum MB1, (ii) evaluation of optimal Leuc. citreum MB1 initial concentration to obtain maximum List. monocytogenes ATCC 15313 inhibition, and (iii) biocontrol of List. monocytogenes ATCC 15313 with Leuc. citreum MB1 during growth in milk at refrigeration temperature. According to our results, it is unquestionable that at least one bacteriocin is active in Leuc. citreum MB1, since important antilisterial activity was verified either in its cell-free supernatant or in co-culture experiments. Co-culture tests showed that ∼107 CFU/ml Leuc. citreum MB1 was the optimal initial concentration to obtain maximum pathogen inhibition. Moreover, Leuc. citreum MB1 was able to delay List. monocytogenes growth at refrigerated temperature.

Characterization of two virulent phages of Lactobacillus plantarum.

We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria.

Streptococcus thermophilus Phages in Whey Derivatives: From Problem to Application in the Dairy Industry.

Fifteen samples of whey protein concentrate (WPC) were tested against 37 commercial Streptococcus thermophilus strains to detect infective bacteriophages. Seventy-three diverse phages were isolated from 12 samples, characterized by using DNA restriction patterns and host range analyses. Sixty-two of them were classified as cos, two as pac, and nine as 5093, according to PCR multiplex assays. Phage concentration was greater than 104 PFU/g for 25.3% of isolated phages. Seven phages showed an unusual wide host range, being able to infect a high number of the tested strains. Regarding thermal resistance, pac phages were the most sensitive, followed by cos phages, those classified as 5093 being the most resistant. Treatments at 85 °C for 5 min in TMG buffer were necessary to completely inactivate all phages. Results demonstrated that the use, without control, of these whey derivatives as additives in dairy fermentations could be a threat because of the potential phage infection of starter strains. In this sense, these phages constitute a pool of new isolates used to improve the phage resistance of starter cultures applied today in the fermentative industry.

Influence of Physicochemical Factors on Adsorption of Ten Shigella flexneri Phages.

Bacterial viruses known as bacteriophages have been demonstrated to be effective in killing foodborne pathogens such as Shigella flexneri. Adsorption is the first step in the phage-host interaction. In the present work, 10 Shigella phages were used to characterize the adsorption process on Shigella flexneri ATCC12022 in several physicochemical conditions related to food and in a food matrix. One-step growth curves were drawn for all the Shigella-phages evaluated. Furthermore, the adsorption rate for each of the 10 phages was determined. In addition, the influence of temperature, Na+, Mg2+, pH, sucrose and glycerol on phage adsorption was investigated. Two phages (Shi22 and Shi30) showed higher burst sizes values (67 and 64 PFU cell-1, respectively) and burst times of 25 min to 30 min, while the other eight phages exhibited burst sizes ranging from 14 to 17 PFU cell-1 with slower burst times. Furthermore, most phages achieved a high adsorption rate, and the adsorption constants (k) ranged from ~10-9 to 10-10 mL min-1. Regarding the influence of temperature, cations and pH, a high or moderate percentage of adsorption was observed for most of the phages evaluated. The adsorption decreased at increasing concentrations of Na+, sucrose and glycerol, although at different levels, since adsorption was more affected by sucrose than by glycerol and Na+ for most phages. The adsorption obtained in Triptein soy broth (TSB) for most of the phages/strain systems evaluated was moderate or high, as well as those observed in a food matrix. Thus, our phages could potentially be used to improve food safety under a wide range of environmental conditions against foodborne pathogens.

Pategrás cheese as a suitable carrier for six probiotic cultures.

The viability of five single-strain and one three-strain probiotic cultures was assessed during Pategrás cheese ripening. Probiotics were inoculated into cheese-milk after a pre-incubation step - intended to improve their survival - or directly as a lyophilised culture; control cheeses without probiotics were also obtained. pH of probiotic and control cheeses was similar, except in probiotic cheeses containing the strain Lb. acidophilus B or the mixed culture. In these cases, the probiotic cheeses were more acid than their respective control cheeses. All the probiotics tested maintained counts above 107 cfu/g during the shelf-life settled for the product. Strains of the Lb. casei group: Lb. paracasei, Lb. casei and Lb. rhamnosus reached and kept the highest cell concentration during cheese ripening, followed by Lb. acidophilus and bifidobacteria. The direct addition of the probiotic cultures was more efficient than their inoculation after a pre-incubation step, for all the probiotics assayed. We have provided evidence that support the use of Pategrás cheese as a performing food-based vehicle for probiotic bacteria.

Thermal, chemical, and photocatalytic inactivation of Lactobacillus plantarum bacteriophages.

The effect of several biocides, thermal treatments, and photocatalysis on the viability of four Lactobacillus plantarum phages was investigated. Times to achieve 99% inactivation (T99) of phages at 63, 72, and 90 degrees C were evaluated in four suspension media: deMan Rogosa Sharpe broth, reconstituted skim milk, a commercial EM-glucose medium, and Tris magnesium gelatin buffer. The four phages studied were highly resistant to 63 degrees C (T99 > 45 min); however, counts < 10 PFU/ml were achieved by heating at 90 degrees C for 5 min. Higher thermal resistance at 72 degrees C was observed when reconstituted skim milk and EM-glucose medium were assayed. Peracetic acid (0.15%, vol/vol) was an effective biocide for the complete inactivation of all phages studied within 5 min of exposure. Sodium hypochlorite (800 ppm) inactivated the phages completely within 30 min. Ethanol (100%) did not destroy phage particles even after 45 min. Isopropanol did not have any effect on phage viability. Phage counts < 50 PFU/ml were obtained within 180 min of photocatalytic treatment. The results obtained in this work are important for establishing adequate methods for inactivating phages in industrial plants and laboratory environments.

Evidence for the presence of restriction/modification systems in Lactobacillus delbrueckii.

The bacteriophages Cb1/204 and Cb1/342 were obtained by induction from the commercial strain Lactobacillus delbrueckii subsp. lactis Cb1, and propagated on Lactobacillus delbrueckii subsp. lactis 204 (Lb.l 204) and Lactobacillus delbrueckii subsp. bulgaricus 342 (Lb.b 342), respectively. By cross sensitivity, it was possible to detect a delay in the lysis of Lb.l 204 with Cb1/342 phage, while the adsorption rate was high (99.5%). Modified and unmodified phages were isolated using phage Cb1/342 and strain Lb.l 204. The EOP (Efficiency of Plaquing) values for the four phages (Cb1/204, Cb1/342, Cb1/342modified and Cb1/342unmodified) suggested that an R/M system modified the original temperate phage, and the BglII-DNA restriction patterns of these phages might point out the presence of a Type II R/M system. Also, the existence of a Type I R/M system was demonstrated by PCR and nucleotide sequence, being the percentages of alignment homology with Type I R/M systems reported previously higher than 95%. In this study it was possible to demonstrate that the native phage resistant mechanisms and the occurrence of prophages in commercial host strains, contribute strongly to diversify the phage population in a factory environment.

Virulence of Leuconostoc phages: Influence of stress conditions associated to dairy processes on their host-phage interactions.

In this work, we assessed the impact of technological cell stress conditions, commonly present in industrial dairy processes, on the host strain-phage interactions in Leuconostoc. Adsorption and burst size of LDG (Leuconostoc pseudomesenteroides) and Ln-9 (Leuconostoc mesenteroides) phages were evaluated under the following conditions: i) MRS broth, 30 °C; ii) MRS broth at pH 5.5, 30 °C (acidic stress); iii) MRS broth added of NaCl at 4% w/v, 30 °C (osmotic stress) and iv) MRS broth, 10 °C (cold stress). Experiences were performed with the host strains growing both in MRS broth (30 °C) and under stress conditions. On the other hand, the effect of diverse levels of NaCl, KCl, saccharose and glucose on the adsorption for LDG phage was evaluated. Acidic and cold conditions did not significantly affect the adsorption rates for any phage. However, adsorption rate of phage LDG was highly reduced under osmotic stress (NaCl), except when the host strain previously grew in presence of the salt. LDG phage adsorption was not modified by addition of saccharides, but it drastically decreased in presence of salts. Acidic conditions did not affect the burst size for LDG phage, but Ln-9 phage diminished this parameter (61 phage particles/infected cell). Latency time showed a lengthening of 10 min for both phages, while the burst time remained unaltered for LDG and it was delayed 10 min for Ln-9. LDG phage did not propagate under osmotic conditions, but Ln-9 phage released phage particles with an important increase of its latent period and burst time. No phage particles were released within 90 min after the adsorption step under cold stress. This is the first report about this subject. Under certain conditions of technological stress (osmotic and cold) associated to dairy processes, phage infections on the two systems studied in this work could be delayed/inhibited.

Inactivation of Dairy Bacteriophages by Thermal and Chemical Treatments.

This article provides information on the characteristics of diverse phages of lactic acid bacteria and highlights the incidence of their presence in different dairy fermentations. As it is known, thermal treatments on raw milk and use of sanitizers in the disinfection of surfaces and equipment are strategies usually applied in dairy to prevent bacteriophage infections. In this sense, this review mainly focuses on the existing data about the resistance against thermal treatments and sanitizers usually used in the dairy industry worldwide, and the differences found among bacteriophages of diverse genera are remarked upon. Also, we provide information concerning the problems that have arisen as a consequence of the potential presence of bacteriophages in cheese whey powder and derivatives when they are added in fermented dairy product manufacturing. Finally, some important conclusions on each topic are marked and checkpoints to be considered are suggested.

New semi-pilot-scale reactor to study the photocatalytic inactivation of phages contained in aerosol.

The aims of this work were to design and build a photocatalytic reactor (UV-A/TiO2) to study the inactivation of phages contained in bioaerosols, which constitute the main dissemination via phages in industrial environments. The reactor is a close system with recirculation that consists of a stainless steel camera (cubic form, side of 60 cm) in which air containing the phage particles circulates and an acrylic compartment with six borosilicate plates covered with TiO2. The reactor is externally illuminated by 20 UV-A lamps. Both compartments are connected by a fan to facilitate the sample circulation. Samples are injected into the camera using two piston nebulizers working in series whereas several methodologies for sampling (impinger/syringe, sampling on photocatalytic plates, and impact of air on slide) were assayed. The reactor setup was carried out using phage B1 (Lactobacillus plantarum), and assays demonstrated a decrease of phage counts of 2.7 log orders after 1 h of photocatalytic treatment. Photonic efficiencies of inactivation were assessed by phage sampling on the photocatalytic plates or by impact of air on a glass slide at the photocatalytic reactor exit. Efficiencies of the same order of magnitude were observed using both sampling methods. This study demonstrated that the designed photocatalytic reactor is effective to inactivate phage B1 (Lb. plantarum) contained in bioaerosols.

Phage-resistant mutants of Lactobacillus delbrueckii may have functional properties that differ from those of parent strains.

Three commercial phage sensitive Lactobacillus delbrueckii strains (identified as Ab(1), YSD V and Ib(3)), and four spontaneous phage-resistant mutants isolated from them were tested for their capacity to activate the gut mucosal immune response in mice, as indicated by the numbers of IgA-producing cells. Random Amplified Polymorphic DNA (RAPD) analysis revealed a strong genetic homology between the sensitive strains and their respective derivatives. The phage-resistant mutants exhibited high levels of phage resistance, elevated stability of this phenotype and technological properties comparable to those of their respective parent strains. The tolerance to acidic conditions, bile salts and lysozyme was strain dependent and total cell viability losses as a result of exposure to all three stresses ranged from 2.0 to 3.7 log units. All the strains were highly resistant to a simulated gastric solution of pH 3, while significant additional losses in cell viability were observed when acid treated cells were exposed to bile salts and lysozyme. BALB/c mice received pure cultures of Lb. delbrueckii sensitive and phage-resistant strains for 2, 5 or 7 consecutive days. The ability of the parent strains to activate the small intestine immune response was preserved or enhanced in phage-resistant mutants. The maximal proliferation of IgA(+) cells was observed at day 5 or 7, depending on the strain. Mutants isolated in this study using natural selection strategies had improved phage resistance, adequate technological properties and satisfactory gut mucosal immunostimulation ability, and so would be good candidates for industrial applications in functional foods.

Spontaneous Lactobacillus delbrueckii phage-resistant mutants with acquired bile tolerance.

Three commercial phage-sensitive strains of Lactobacillus delbrueckii (strains Ab(1), YSD V and Ib(3)) and four spontaneous phage-resistant mutants (strains A(7), A(17), V(2) and I(39)) isolated from them, all with a probiotic potential previously demonstrated were studied for their tolerance of bile salts (ox gall). Minimal Inhibitory Concentrations (MICs) ranged from 0.30% to 0.35% (w/v) of ox gall. These strains were exposed to gradually increasing concentrations of ox gall with the aim of isolating bile resistant derivatives. Stable derivatives able to tolerate up to 0.9% of ox gall were obtained from L. delbrueckii Ab(1), as well as from its spontaneous phage-resistant mutants A(7) and A(17). Random Amplified Polymorphic DNA (RAPD-PCR) analysis revealed a strong genetic homology between the ox gall-tolerant derivatives and their respective non-adapted original strains. These derivatives maintained, in general, the phage resistance phenotype of the non-adapted strains, with only one exception (phage-resistant mutant A(7)). After progressive ox gall adaptation, the phage-resistant mutant A(7) also exhibited progressive reversion of the phage resistance phenotype. The derivative with the highest ox gall-acquired tolerance (A(7)(0.9)) became sensitive to the phage, but derivatives with low (A(7)(0.3)) and intermediate (A(7)(0.6)) ox gall-acquired tolerance retained phage resistance. The technological properties of ox gall derivatives were comparable to those of their respective parent strains. However, the cells of the former were smaller than those of the original strains. Finally, the tolerant derivatives grew faster in the presence of ox gall than the parent strains. Our results demonstrated that it was possible to obtain, by a natural selection strategy, probiotic strains with acquired ox gall-tolerance from three (L. delbrueckii Ab(1) and their phage-resistant mutants A(7) and A(17)) of seven tested strains. Since such derivatives keep both phage resistance and other useful technological properties, they could be used for production of functional foods.

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.