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.

Bacteriophages in dairy plants.

Bacteriophages represent the main microbiological threat for the manufacture of fermented foods. The dairy industry is the most affected by this problem, as phages are naturally present in raw milk, surfaces, vats, tanks, floors, and distributed by air displacements. Cheese whey may also contain high phage concentrations. Prophages harbored by lysogenic strains could be induced, generating new lytic phages. In this context, where phages cannot be eradicated from dairies, methods of phage monitoring are mandatory. These are mainly based in microbiological features, like classical methods, that are the most used, economic and simple to carry out. Phage DNA detection and quantification by PCR and qPCR, more complex and expensive, are faster, although not able to discern between viable and non-viable virions. Electron microscopy allows direct visualization and characterization of phage morphology, but the apparatus is expensive. Alternative methods based in other phage traits also exist, though less studied and not applicable on a daily basis. Recognition of contamination sources and correct phage monitoring in dairy factories allow a correct application of control measures. These include general measures such as proper factory design, efficient programs of sanitization, good treatment of raw materials, especially milk, and careful handling of by-products. Additionally, the use of starts cultures should be adequate, with application of rotation schemes when possible. Finally, the selection of bacteriophage insensitive mutants (BIM) is essential, and can be achieved simply and empirically, though the study of CRISPR-Cas and other newly discovered mechanisms provide a more rational basis to obtain BIMs with optimized features.

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.

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 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 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.

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.