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.Fil: Briggiler Marcó, Mariángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Reinheimer, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Quiberoni, Andrea del Lujan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; Argentin

Estudio de la inactivación fotocatalítica de fagos de bacterias lácticas en un reactor a escala semi-piloto

Las infecciones fágicas continúan siendo una amenaza para los procesos fermentativos, a pesar de las diversas estrategias implementadas a nivel industrial para disminuir su incidencia. El objetivo del presente trabajo fue investigar la inactivación de fagos de bacterias lácticas, contenidos en el aire, en un reactor fotocatalítico (TiO2 como catalizador, UV-A) a escala semi-piloto, ajustando previamente diversas condiciones de operación. En este sentido, utilizando el fago B1 (infectivo de Lactobacillus plantarum ATCC 8014) se ajustó el método de preparación de la suspensión fágica (concentración por centrifugación en tubos concentradores Vivaspin, agregando o no una etapa previa de concentración con PEG 8000, 10% m/v), de nebulización (utilizando 2 nebulizadores a pistón trabajando en serie o el nebulizador Collison 6-jet) así como también la influencia de la humedad relativa en el interior del reactor (73 y 46%). Posteriormente, los ensayos de inactivación fotocatalítica involucraron a los fagos B1 y LDG y Ln9 infectivos de las cepas Lb. plantarum ATCC 8014, Leuconostoc pseudomesenteroides R707 y Leuconostoc mesenteroides 79-1, respectivamente. Para estos ensayos, las suspensiones fágicas fueron diluidas (1:10) en agua destilada estéril y nebulizadas en el interior de la cámara (durante 30 min), llevando a cabo los ensayos de inactivación como función del tiempo durante 100 min (tiempo total). Según los resultados obtenidos, basados en una mejor {performance} del fago B1 contenido en el aire en el interior del reactor, se seleccionaron como condiciones de operación, la concentración de la suspensión fágica mediante tubos concentradores Vivaspin (sin etapa previa de concentración con PEG), la utilización del nebulizador Collison y una humedad relativa de 73% en el interior del equipo. Con respecto a los ensayos de inactivación, los títulos obtenidos mientras el nebulizador estuvo funcionando (30 min) fueron de 2,0x10^4, 5,0 x10^3 y de 3,0 x10^3 UFP/ml para los fagos B1, LDG y Ln9, respectivamente. Cuando el proceso de nebulización finalizó, se observó una disminución en los títulos fágicos tanto en presencia como en ausencia de radiación UV-A (y presencia de catalizador). En este sentido, en ausencia de radiación UV, se observó una disminución de 2 órdenes log a los 100 min de experiencia para los 3 fagos mientras que en presencia de luz UV se logró su inactivación completa (títulos < 10 UFP/ml) dentro de los 80 min (B1) y 100 min de tratamiento fotocatalítico (LDG y Ln9). El reactor fotocatalítico a escala semi-piloto resulta efectivo para la inactivación de fagos de bacterias lácticas contenidos en bioaerosoles. Esta tecnología podría implementarse en conjunto con otras estrategias para disminuir el riesgo de infecciones fágicas en los ambientes industriales.Fil: Gornati, Jesica Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Negro, Antonio Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Briggiler Marcó, Mariángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaXXII Encuentro de Jóvenes InvestigadoresSanta FeArgentinaUniversidad Nacional del Litora

Thermal, Chemical, and Photocatalytic Inactivation of <i>Lactobacillus plantarum</i> 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.Centro de Investigación y Desarrollo en Criotecnología de Alimento

Thermal, Chemical, and Photocatalytic Inactivation of <i>Lactobacillus plantarum</i> 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.Centro de Investigación y Desarrollo en Criotecnología de Alimento

Bacteriocins from lactic acid bacteria: strategies for the bioprotection of dairy foods

Lactic acid bacteria (LAB), essential in fermenting milk, produce an array of antimicrobial compounds, notably bacteriocins, which contribute to the extended shelf life of dairy goods. Bacteriocins, ribosomally synthesized peptides, display broad or narrow-spectrum antimicrobial activity, thus holding promise in food preservation. The classification of LAB bacteriocins is intricate, reflecting evolving genomic insights and biosynthesis mechanisms. Strategies for integrating bacteriocins into dairy products include purified forms, bacteriocin-producing LAB, and bacteriocin-containing fermentates, each with distinct advantages and considerations. Optimization of fermentation conditions, encompassing time, temperature, pH, and culture medium, is essential for maximizing bacteriocin production. This optimization facilitates enhanced quality and safety of fermented dairy items, aligning with the growing consumer preference for natural, minimally processed foods. Furthermore, the incorporation of bacteriocins into a hurdle approach alongside thermal and non-thermal treatments holds promise for augmenting food bioprotection while reducing reliance on chemical preservatives. This comprehensive overview underscores the potential of LAB bacteriocins as a natural, effective alternative to conventional food preservatives, offering insights into their application and optimization in dairy product preservation

Phage infections in Lactobacillus plantarum. Characterization and industrial implications

Dos fagos de colección de Lactobacillus plantarum (ATCC 8014-B1 y ATCC 8014-B2) fueron sometidos a estudios moleculares que incluyeron la determinación del mecanismo de empaquetamiento del ADN fágico, el secuenciamiento de los genomas y la identificación de sus proteínas estructurales. Adicionalmente, se estudió la morfología de ambos fagos. Por otro lado, se evaluó la viabilidad durante la conservación a diversas temperaturas y valores de pH de los fagos de colección y de dos fagos aislados previamente de gránulos de kefir (FAGK1 y FAGK2). Posteriormente, se caracterizó la interacción fago-cepa: influencia de cationes divalentes en el ciclo lítico, rango de hospedadores, ciclos de multiplicación fágica, proceso de adsorción, receptores fágicos y el mecanismo de fagorresistencia del tipo Restricción/Modificación. En el segundo capítulo de esta tesis, se estudió el comportamiento de los fagos frente a diferentes estrategias que podrían utilizarse para disminuir el riesgo de infecciones fágicas. Esto incluyó el estudio de la resistencia fágica a tratamientos térmicos, químicos y fotocatalíticos, así como también el aislamiento de mutantes espontáneos fagorresistentes con aptitudes tecnológicas. En particular, la eficiencia de la fotocatálisis en la inactivación fágica fue evaluada para fagos de diversas especies de bacterias lácticas. Los resultados obtenidos en este estudio permiten profundizar los conocimientos referidos a infecciones fágicas en Lb. plantarum, los cuales serían necesarios si esta especie bacteriana es utilizada en el desarrollo de alimentos funcionales. Por otro lado, este trabajo demostró que es posible el aislamiento de variantes fagorresistentes con adecuadas propiedades tecnológicas.In this Thesis, a molecular characterization (including DNA packaging mechanisms, genome sequencing and structural protein identification) of two Lactobacillus plantarum collection phages (ATCC 8014-B1 and ATCC 8014-B2) was carried out. In addition, the phage morphology was studied. On the other hand, phage viability throughout storage at different pH and temperature was determined for collection phages and for phages FAGK1 and FAGK2 (isolated from kefir grains). To characterize the interaction between phages and their sensitive strains, the following aspects were studied: influence of divalent cations during the lytic cycle, host spectrum, one step growth curves, characterization of the adsorption step, phage receptors and presence of restriction-modification (R/M) systems. In the second chapter of this Thesis, the behavior of phages subjected to thermal, chemical and photocatalytic treatments, used to diminish the frequency of phage infections, was studied. In particular, the efficiency of photocatalysis on phage inactivation was studied for phages infecting diverse species of lactic acid bacteria. Also, the isolation of phage-resistant mutants with adequate technological properties was carried out. Results obtained in this Thesis significantly contributed to improve the knowledge about phage infections on Lb. plantarum, which would be useful if this bacterial species is used in the manufacture of functional foods. On the other hand, this work demonstrated that the assayed methodologies are efficient for obtaining spontaneous phage-resistant mutants from Lb. plantarum strains with adequate technological properties.Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) American Society for Microbiology (ASM

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 &deg;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.Fil: Briggiler Marcó, Mariángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Mercanti, Diego Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; Argentin

Bacteriophages and dairy fermentations

This review highlights the main strategies available to control phage infection during large-scale milk fermentation by lactic acid bacteria. The topics that are emphasized include the factors infuencing bacterial activities, the sources of phage contamination, the methods available to detect and quantify phages, as well as practical solutions to limit phage dispersion through an adapted factory design, the control of air fow, the use of adequate sanitizers, the restricted used of recycled products, and the selection and growth of bacterial cultures