Bacteriophages on dairy foods

This review focuses on the impact of bacteriophages on the manufacture of dairy foods. Firstly, the impact of phages of lactic acid bacteria in the dairy industry, where they are considered enemies, is discussed. The sources of phage contamination in dairy plants are detailed, with special emphasis on the rise of phage infections related to the growing use of cheese whey as ingredient. Other topics include traditional and new methods of phage detection, quantification and monitoring, and strategies of phage control in dairy plants, either of physical, chemical or biological nature. Finally, the use of phages or purified phage enzymes as allies to control pathogenic bacteria in the food industry is reviewed.Fil: Pujato, Silvina. 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; 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

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

Lactobacillus casei/paracasei: aislamiento de mutantes espontáneos fagorresistentes para uso industrial

ResumenLos fagos de bacterias probióticas son causa de enormes pérdidas económicas y severas limitaciones a las estrategias de control. En este trabajo se aislaron y caracterizaron mutantes de L. casei/paracasei empleando el primer fago L. paracasei aislado en Sudamérica en una planta industrial argentina. Se caracterizaron fenotipo (eficiencias de recuperación de resistentes verdaderos, estabilidad, resistencia frente a otros fagos específicos) y mecanismos de fagorresistencia (nivel de fagorresistencia, liberación espontánea de fagos virulentos, adsorción con y sin calcio). Se demostró que el aislamiento de mutantes espontáneos resistentes desde bacterias probióticas sensibles, resulta una alternativa válida para obtener cepas para rotar en procesos industriales. Summary Phage attacks to probiotic strains cause enormous economic losses and limit common control strategies. In this work, spontaneous bacteriophage insensitive mutants were isolated from sensitive L. casei/paracasei strains against the first Sudamerican L. paracasei phage isolated in an Argentinean dairy plant. Phage resistance phenotype (efficiency of phage-resistant recovery, stability, sensitivity against other specific phages) and phage resistance mechanisms (phage resistance level, spontaneous release of virulent phages, phage adsorption with or without cacium ions) were characterized. The isolation of spontaneous phage-resistant mutants, from sensitive probiotic bacteria, was demonstrated to be a useful tool to obtain strains to rotate in industrial processes.Fil: Capra, María Luján. 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; 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

High pressure homogenization versus heat treatment: effect on survival, growth, and metabolism of dairy Leuconostoc strains.

The effect of high pressure homogenization (HPH) with respect to a traditional heat treatment on the inactivation, growth at 8°C after treatments, and volatile profile of adventitious Leuconostoc strains isolated from Cremoso Argentino spoiled cheeses and ingredients used for their manufacture was evaluated. Most Leuconostoc strains revealed elevated resistance to HPH (eight passes, 100 MPa), especially when resuspended in skim milk. Heat treatment was more efficient than HPH in inactivating Leuconostoc cells at the three initial levels tested. The levels of alcohols and sulfur compounds increased during incubation at 8°C in HPH-treated samples, while the highest amounts of aldehydes and ketones characterized were in heated samples. Leuconostoc cells resuspended in skim milk and subjected to one single-pass HPH treatment using an industrial-scale machine showed remarkable reductions in viable cell counts only when 300 and 400 MPa were applied. However, the cell counts of treated samples rose rapidly after only 5 days of storage at 8°C. The Leuconostoc strains tested in this work were highly resistant to the inactivation treatments applied. Neither HPH nor heat treatment assured their total destruction, even though they were more sensitive to the thermal treatment. To enhance the inhibitory effect on Leuconostoc cells, HPH should be combined with a mild heat treatment, which in addition to efficient microbial inactivation, could allow maximal retention of the physicochemical properties of the product

Phage resistance of acid lactic bacteria isolated from Coalho cheese industries

Este trabalho teve como objetivos isolar bacteriófagos de amostras de leite, soro e queijo de Coalho e avaliar a resistência de cepas de Lactobacillus paracasei, pertencentes à Coleção de Micro-organismos de Interesse para a Agroindústria Tropical da Embrapa Agroindústria Tropical, aos fagos isolados. Posteriormente, a resistência destas cepas a fagos específicos para L. paracasei, da Coleção do Instituto de Lactología Industrial - INLAIN (Santa Fe, Argentina), também foi avaliada. As amostras para isolamento dos fagos foram obtidas em quatro unidades de processamento de queijo de Coalho, sendo duas artesanais e duas industriais, localizadas no Estado do Ceará. Para o isolamento dos bacteriófagos, foi empregado o teste de lise celular (spot), enquanto que a resistência das culturas aos fagos foi avaliada pelos testes de capacidade de produção de ácido e avaliação da turbidez. As cepas avaliadas foram resistentes aos bacteriófagos provenientes das unidades de processamento de queijo de Coalho e aos bacteriófagos da Coleção do INLAIN. Os resultados obtidos indicaram que as culturas láticas testadas, resistentes aos bacteriófagos, podem ser utilizadas na composição de fermento lático destinado à elaboração de queijo de Coalho, a partir de leite pasteurizado.The objectives of this research were to isolate bacteriophages from milk samples, whey and Coalho cheese and to evaluate the resistance of strains of Lactobacillus paracasei from the Collection of Microorganisms of Interest for the Tropical Agroindustry, belonging to Embrapa Tropical Agroindustry, to isolate phages. The strains resistance to specific L. paracasei phages from the collection of the Instituto de Lactolog a Industrial - INLAIN (Santa Fe, Argentina) was also evaluated. Samples for phage isolation were from four Coalho cheese processing units, two artisanal and two industrial, localized in the state of Ceara. Spot test was employed for bacteriophages isolation, while the culture phage resistance was evaluated by the acid production and turbidity tests. The strains were resistant both to phages isolated from Coalho cheese processing units and phages from collection of INLAIN. The results showed that lactic cultures evaluated here were resistant to bacteriophages and they can be used in the composition of lactic cultures specifically for Coalho cheese manufacture from pasteurized milk

Descontaminación de superficies sólidas mediante cócteles fágicos para prevenir la contaminación cruzada por Escherichia coli enteropatógena y shigatoxigénica

El biocontrol de cepas de Escherichia coli enteropatógena (EPEC) y Shigatoxigénica (STEC), causantes de enfermedades trasmitidas por alimentos, mediado por fagos puede ser una herramienta interesante de control para evitar la contaminación cruzada tanto en la industria como en el hogar. Con el objetivo de reducir la contaminación por parte de bacterias patógenas nos propusimos realizar ensayos para evaluar el nivel de biocontrol en medio líquido (challenge en caldo Hershey + MgSO4 5 mM, Hershey-Mg) tanto con fagos individuales como formando parte de un cóctel así como la descontaminación de superficies sólidas, a saber acero inoxidables (AI) (25 mm x 15 mm) y cubreobjetos de vidrio (CV) (18 mm x 18 mm), mediante cócteles fágicos a 4 y 37 ºC. En los challenge se ensayaron 100 µl de fago sobre 5 ml de cultivo bacteriano (DO600nm = 0,1), posteriormente se realizaron recuentos bacterianos a 2, 6 y 24 h. El fago DT1 se ensayó sobre las cepas EPEC920 (eaeA+) (Multiplicidad de infección, MOI = 8,6x101) y STEC O157:H7 (464) (stx2+ y eaeA+) (MOI = 1,4x10exp2), el DT5 sobre STEC no-O157 (ARG4827) (stx1+ y stx2+) (MOI = 2,9x10exp2) y DT6 sobre las tres cepas mencionadas anteriormente a una MOI de 7,8x10exp1, 1,3x10exp2 y 2,6x10exp2, respectivamente. En los ensayos sobre superficies sólidas, las matrices se limpiaron con etanol al 70 % y se autoclavaron. Cada bacteria se inoculó a dos concentraciones, a saber aproximadamente 10exp4 y 10exp6 UFC/ml para CV y 10exp5 y 10exp7 UFC/ml para AI, se dejó secar a temperatura ambiente, se aplicó 100 µl del cóctel de fagos (109 UFP/ml) correspondiente [(DT1 + DT6 para EPEC920 y STEC O157:H7 (464) y DT5 + DT6 para STEC no-O157 (ARG4827)], o caldo Hershey-Mg (control) y se incubó a 4 y 37 ºC durante 1, 3 y 24 h. Pasado el tiempo de incubación, las matrices se procesan para la extracción y recuento de células bacterianas. En los challenge, tanto con fagos individuales como con el cóctel obtuvimos reducciones significativas de células viables y un comportamiento similar para todas las cepas evaluadas a 37 ºC, mientras que a 4 ºC, si bien en la mayoría de los casos se obtuvieron reducciones significativas, éstas fueron pequeñas. En CV y AI el cóctel de fagos inactivó rápida y completamente tanto a la cepa EPEC920 como a STEC O157:H7 (464) a las temperaturas (4 y 37 ºC) y MOI evaluadas. Sin embargo, se observó un recrecimiento cuando se empleo MOI baja para STEC O157:H7 (464) a 4 y 37 ºC en CV y AI, respectivamente, así como para EPEC920 a 37 ºC a ambos valores de MOI empleados. En CV, STEC no-O157 (ARG4827) fue significativamente reducida por el cóctel solo a las primeras horas del ensayo tanto a 5 como a 37 ºC, sin embargo, no se obtuvo una inactivación total del patógeno y se observó un recrecimiento a 37 ºC. Contrariamente, en AI se obtuvo una inactivación completa de STEC no-O157 (ARG4827) a ambas temperaturas evaluadas. En base a nuestros resultados los bacteriofagos resultan promisorios para el biocontrol de cepas patógenas de E. coli, sin embargo, es necesario optimizar cada sistema fago/cepa para prevenir, debido al recrecimiento observado en algunos casos, la potencial aparición de resistencia.Fil: Tomat, David Damian. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Microbiologia; ArgentinaFil: Quiberoni, Andrea del Lujan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Lactologia Industrial; ArgentinaFil: Balagué, Claudia. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Microbiologia; Argentin

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 to #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-codingregion (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). Detectionof 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 methodsuccessfully detected and discriminated groups of prophages or remnants in L. casei group strains.Fil: Zaburlin, Delfina. 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; 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

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 influencing 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 flow, the use of adequate sanitizers, the restricted used of recycled products, and the selection and growth of bacterial cultures.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: Moineau, Sylvain. Université Laval; CanadáFil: 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

Technological performance of spontaneous phage-resistant derivatives of Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides during milk fermentation

A total of 142 presumptive phage-resistant derivatives were isolated from six commercial phage-sensitive Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides strains, using six phages either individually or in combination (cocktail). Genetic diversity, efficiency of plaquing (EOP), phage-resistance stability, lysogeny, adsorption rates and technological performance were determined for the 89 confirmed phage-resistant variants. Derivatives showed very low EOP values (<10−10) and high stability of phage-resistance phenotype. Some mutants showed low adsorption rates (10–42%) thus indicating adsorption interferences. Additional resistance mechanisms (operative at later stages) were suggested for mutants revealing high phage adsorption rates. A good performance during milk fermentation and the subsequent refrigerated storage in the presence of phages was demonstrated for four mutants, selected on the basis of their stability and low or null phage adsorption. These phage-resistant variants could be used in industrial rotation schemes when commercial strains become sensitive to phages present in the environment.Fil: Pujato, Silvina. 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; ArgentinaFil: Guglielmotti, Daniela Marta. 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