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.

Phage adsorption to Lactobacillus plantarum: influence of physiological and environmental factors.

Bacteriophage infection of lactic acid bacteria (LAB) constitutes one of the major problems in the dairy industry, causing economic losses and a constant risk of low quality and/or unsafe foods. The first step in the phage biology is the adsorption on the host cell surface. In a previous study, a remarkable thermal, chemical and photocatalytic resistance was demonstrated by four phages of Lactobacillus plantarum (ATCC 8014-B1, ATCC 8014-B2, FAGK1 and FAGK2). In the present work, these phages were used to characterize the adsorption process on L. plantarum ATCC 8014. Clearly, the characterization of this process could increase the possibilities of design useful strategies in order to prevent phage infections. The influence of Ca(2+), temperature, pH and physiological cell state on phage adsorption was investigated. Burst sizes of phages ATCC 8014-B1 and ATCC 8014-B2 were 60 and 83 PFU/infective centre, respectively. The four phages exhibited a high infectivity even at pH 4 and pH 11. Calcium or magnesium ions were not indispensable for cell lysis and plaque formation, and more than 99% of phage particles were adsorbed either in the presence or absence of Ca(2+), after 15 min at 37 degrees C. Phage adsorption was only partially affected at 50 degrees C, while reached its maximum between 30 and 42 degrees C. The highest adsorption values (99.9%) were observed from pH 5 to 7, after 30 min at 37 degrees C. Adsorption rates decreased after the thermal inactivation of cells, though, when 20 microg/ml of chloramphenicol was used, adsorption values were similar on treated and untreated cells. All these results showed that the adsorption process was only partially affected by a few conditions: thermally killed host cells, an incubation temperature of 50 degrees C and pH values of 9 and 10. Nevertheless, and unfortunately, those conditions are not commonly applied during fermented food manufacturing, thus restricting highly the application of strategies currently available to reduce phage infections in industrial environments. This work also contributes to increase the currently knowledge on the biological aspects of L. plantarum bacteriophages.

Lactobacillus plantarum bacteriophages isolated from Kefir grains: phenotypic and molecular characterization.

Two greatly related Lactobacillus plantarum bacteriophages (named FAGK1 and FAGK2) were isolated from Kefir grains of different origins. Both phages belonged to the Siphoviridae family (morphotype B1) and showed similar dimensions for head and tail sizes. The host range of the two phages, using 36 strains as potential host strains, differed only in the phage reactivity against one of them. The phages showed latent periods of 30 min, burst periods of 80+/-10 min and burst size values of 11.0+/-1.0 PFU per infected cell as mean value. Identical DNA restriction patterns were obtained for both phages with PvuI, SalI, HindIII and MluI. The viral DNA apparently did not present extremes cos and the structural protein patterns presented four major bands (32.9, 35.7, 43.0 and 66.2 kDa). This study reports the first isolation of bacteriophages of Lb. plantarum from Kefir grains and adds further knowledge regarding the complex microbial community of this fermented milk.

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.