Encapsulation of specific Salmonella Enteritidis phage f3αSE on alginate-spheres as a method for protection and dosification

Background: Bacteriophages have been proposed as an alternative to control pathogenic bacteria resistant to antibiotics. However, they are not extensively used due to different factors such as vulnerability under environmental conditions and the lack of efficient administration methods. A potential solution is the encapsulation of bacteriophages in hydrogel polymers to increase their viability and as a controlled release method. This work describes the use of alginate-Ca+2 matrixes as mechanisms for protection and dosification of the phage f3αSE which has been successfully used to prevent infections produced by Salmonella Enteritidis. Results: The viability of the pure phage is reduced in near 100% after 1-h incubation at pH 2 or 3. However, the encapsulated phage remains active in 80, 6% at pH 3, while no differences were observed at pH 2, 4 or 7. Exposition of f3αSE to different T° showed that the viability of this phage decreased with increased T° to near 15% at 60°C, while the encapsulated phage remains with 50% viability at same temperature. Finally, the encapsulation of phages showed to extend their presence for 100 h in the medium compared to non-encapsulated phages in a water flow system, which simulate automatic birdbath used in poultry industry, maintaining the phage concentration between 102 and 104 PFU/mL during 250 h. Conclusions: Encapsulation in alginate-Ca+2 spheres can be a good alternative to extend viability of phages and can be used as a phage method dosification method in water flow systems.

Bacteriophages in the control of pathogenic vibrios

Vibrios are common inhabitants of marine and estuarine environments. Some of them can be pathogenic to humans and/or marine animals using a broad repertory of virulence factors. Lately, several reports have indicated that the incidence of Vibrio infections in humans is rising and also in animals constitute a continuing threat for aquaculture. Moreover, the continuous use of antibiotics has been accompanied by an emergence of antibiotic resistance in Vibrio species, implying a necessity for efficient treatments. One promising alternative that emerges is the use of lytic bacteriophages; however, there are some drawbacks that should be overcome to make phage therapy a widely accepted method. In this work, we discuss about the major pathogenic Vibrio species and the progress, benefits and disadvantages that have been detected during the experimental use of bacteriophages to their control.

JMT-1: a novel, spherical lytic halotolerant phage isolated from Yuncheng saline lake

ABSTRACT This work described a novel halotolerant phage, JMT-1, with a spherical morphology. JMT-1, which was isolated from a hypersaline lake, could produce clear plaques on Chromohalobacter sp. LY7-3. The purified virions are spherical, have no visible tail, and are about 30-50 nm in diameter. JMT-1 has a wide host range, and this study showed that the phage can infect at least five halophilic bacteria. The proteins of JMT-1 were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and six proteins were detected. Results show that JMT-1 is a bacteriophage with a linear double-stranded DNA. Meanwhile, the genome is approximately 23 kb in length and is sensitive to the restriction endonucleases Bam I, EcoR I, Hind III and Kpa I. JMT-1 has a high titer, approaching 1.5 × 109 pfu/mL after dilution to 10−6 pfu/mL. The phage is also sensitive to chloroform but not to temperature, pH, and lowered salt concentration. JMT-1 is a spherical lytic halotolerant phage with a wide host range and has the tolerance to specific extreme environments. These data could provide references for studying phage resources in extreme environments and would also provide the useful methods for isolation and identification of other valuable phage in the salt lake environment.

Antibacterial efficacy of nisin, bacteriophage P100 and sodium lactate against Listeria monocytogenes in ready-to-eat sliced pork ham

ABSTRACT The effectiveness of bacteriophage P100, nisin and sodium lactate, individually and in combination, in inhibiting Listeria monocytogenes in ready-to-eat pork ham slices was assessed. The antimicrobials were applied to the surfaces of ready-to-eat pork ham slices, which were inoculated with a mixture of L. monocytogenes. Among the individual antimicrobial treatments, bacteriophage P100 was the most effective, decreasing L. monocytogenes to undetectable levels at zero and 72 h post-infection. Sodium lactate was the least effective treatment. Treatment with nisin at zero h significantly reduced initial cell density (p < 0.05). However, this pattern was not observed at 72 h of storage. A significant difference (p < 0.05) existed between the results of separate bacteriophage and nisin treatments after refrigerated storage, but not immediately upon inoculation of the bacteria. The results showed that the use of bacteriophage P100 is the method of choice for the control of bacteria.

El desafío de controlar las enfermedades transmitidas por alimentos: bacteriófagos como una nueva herramienta biotecnológica / The challenge of controlling foodborne diseases: bacteriophages as a new biotechnological tool

Foodborne diseases are an increasing public health issue, in which bacterial pathogens have a transcendental role. To face this situation, the food industry has implemented several control strategies, using in the last decade some biotechnological tools, such as direct application of bacteriophages on food, to effectively control bacterial pathogens. Their bactericidal and safe properties to humans and animals have been widely described in the literature, being nowadays some bacteriophage-based products commercially available. Despite this, there are so many factors that can interfere in their biocontrol effectiveness on food, therefore is essential to consider these factors before their application. Thus, the optimal bacterial reduction will be achieved, which would produce a safer food. This review discusses some factors to consider in the use of bacteriophages as biocontrol agents of foodborne pathogens, including historical background, taxonomy and biological description of bacteriophages, and also advantages, disadvantages, and considerations of food applications.

Las enfermedades transmitidas por alimentos son un creciente problema de salud pública, donde los agentes patógenos bacterianos juegan un rol trascendental. La industria alimentaria ha implementado diversas medidas de control para enfrentar esta situación, utilizando en la última década algunas herramientas biotecnológicas, como es la aplicación de bacteriófagos directamente en los alimentos. Sus propiedades exclusivamente bactericidas e inocuas para el hombre y los animales han sido descritas ampliamente en la literatura científica, existiendo a la fecha algunos productos comerciales disponibles en el mercado internacional. A pesar de esto, diversos son los factores que pueden influir en su efectividad bio-controladora en alimentos, por lo que conocer dichos factores resulta fundamental antes de considerar su aplicación. De esta manera, se logrará obtener la máxima actividad reductora de la carga bacteriana, generando así un alimento más seguro. Esta revisión aborda ciertos factores a considerar para el uso de bacteriófagos como agentes bio-controladores de patógenos alimentarios, incluyendo antecedentes históricos, taxonomía y descripción biológica de bacteriófagos, así como ventajas, desventajas y consideraciones de su aplicación en alimentos.

Liquid based formulations of bacteriophages for the management of waterborne bacterial pathogens in water microcosms.

Water resources are contaminated by life-threatening multidrug resistant pathogenic bacteria. Unfortunately, these pathogenic bacteria do not respond to the traditional water purification methods. Therefore, there is a need of environmentally friendly strategies to overcome the problems associated with the antimicrobial resistant bacterial pathogens. In the present study, highly potent lytic phages against multidrug-resistant Salmonella enterica serovar Paratyphi B, Pseudomonas aeruginosa and Klebsiella pneumoniae were isolated from the Pavana river water. They belonged to the Podoviridae and Siphoviridae families. These phages were purified and enriched in the laboratory. Monovalent formulations of φSPB, BVPaP-3 and KPP phages were prepared in three different liquids viz., phage broth, saline and distilled water. The phages were stable for almost 8-10 months in the phage broth at 4 °C. The stability of the phages in saline and distilled water was 5-6 months at 4 °C. All of the phages were stable only for 4-6 months in the phage broth at 30 °C. The monovalent phage formulation of φSPB was applied at MOI < 1, as disinfectant against an exponential and stationary phase cells of Salmonella enterica serovar Paratyphi B in various water microcosms. The results indicated that there was almost 80 % reduction in the log phase cells of Salmonella serovar Paratyphi B in 24 h. In stationary phase cells, the reduction was comparatively less within same period. At the same time, there was concomitant increase in the phage population by 80% in all the microcosms indicating that φSPB phage is highly potent in killing pathogen in water. Results strongly support that the formulation of φSPB in the phage broth in monovalent form could be used as an effective biological disinfectant for preventing transmission of water- borne bacterial pathogens, including antimicrobial resistant ones.

Estabilidad de fagos de Lactococcus lactis frente al hipoclorito de sodio y durante el almacenamiento / Stability of Lactococcus lactis phages treated with sodium hypochlorite and during storage

Survival of lytic bacteriophages active against Lactococcus lactis ssp. lactis and ssp. cremoris was determined after treatment with sodium hypochlorite and during storage at 4 degrees C. Three phages were isolated from dairy plants in Argentina (ARG) and the other phages were isolated in the United States of America (US). All of them represent phages that infected cheese manufacture industries and belong to different morphological or serological groups. These phages showed higher survival in M17 broth, buffered with sodium glycerophosphate, than in trypteine soy broth (TSB). Phage populations did not decrease significantly during 14 weeks in M17 broth, whereas in TSB the titers of phage suspensions began to decline around 9 days. In addition, the effect of sodium hypochlorite was more marked in broth than in milk. A higher surviving fraction was obtained in milk, even when tenfold higher concentrations of chlorine were used. The effect of hypochlorite on phages of the same serological group was quite similar and independent of phage morphology. However, phage 137-1, which belongs to other serological group, showed lower resistance to sodium hypochlorite. Comparing the hypochlorite inactivation for ARG and US phages, it was observed that they have their own inactivation values, independently of their origin and morphological group. Long periods of time and high concentrations of chlorine were necessary to reduce the surviving fraction in milk. This indicates that hypochlorite concentrations and times of contact can be critical for the efficiency of the operative sanitization processes.

Biological activity & interaction of Vibrio cholerae bacteriophages in rabbit ileal loop.

A set of ten V. cholerae EITor phages is in routine use for phage typing of V. cholerae O1 biotype EITor strains. These phages were used in rabbit ileal loop experiment to investigate whether these phages have any prophylactic value as regards their lytic capability on V. cholerae strains. The phages were found to have no prophylactic use as they were unable to lyse the standard bacterial strain V. cholerae MAK 757.

Partial characterization of bacteriophages active against rhizobium leguminosarum Isolated from some Egyptian soils

Seventeen rhizobiophages infective against Rhizobium leguminosarwn were isolated from field grown Vicia faba in AI-Ibrahimia [Sharkia Governorate-Egypt]. Morphology, host range and inactivation pattern of the phage to heat and UV-light were studied. Isolated phages had isometric heads and tails which were contractile [2 phages], long and non contractile [7 phages], or very short tails [8 phages]. The phages were members of Bradley's basic morphological groups A, B and C. Most of these phages showed, specificity for their host [R. leguminosarune local strain] and distinct rates of sensitivity against heat and UV

Mutante de Streptococcus lactis con resistencia a bacteriófagos aislados en Argentina y Estados Unidos de América / A mutant of Streptococcus lactis with resistance to bacteriophages isolated in Argentina and the United States of America

Streptococcuslactis y S. cremoris, bacterias lácticas utilizadas para la producción de quesos, pueden reesultar infectadas por bacteriófago específicos, lo que ocasiona alteraciones en la calidad, disminución de la productividad económicas significativas. Los métodos de desinfección y control más usuales no siempre resultan en una completa protección contra la proliferación de los fagos, por los que se ha recurrido al empleo de cepas resistentes a los mismos. En el presente trabajo se ha empleado un método simple y directo para obtener mutantes espontáneas de S. lactis con resistencia a fagos. La mutante S.lacts C26 obtenida a partir de la cepa S. lactis C2 sensible a fagos exhibe propiedades bioquímicas y microbiológicas similares a las de la cepa original C2, diferenciándose de ella por una clara resistencia al fago St15, de morfología alargada, utilizado para la selección. Las curvas de la cinética de crecimiento de la mutante en ausencia y en presencia del fago resultaron semejantes. La cepa C26 fue controlada con otros dos fagos aislados en Argentina y con seis fagos de dos tipos morfológicos, aislados en la Universidad de Cornell en EE.UU. La cepa C26 que fuera aislada como mutante resistente al fafo St15, resultó también resistente a todos ellos. Por otra parte, ha sido de interés verficar que los fagos no siempre observan una especificidad estricta con respecto a la especie de la célula huésped. El fago D59-1, aislado utilizando una cepa S. cremoris, también provoca una lisis significativa en la cepa S. lactis C2, suiriendo que ambas poseen sitios receptores semejantes. La mutante C26 fue empleada con éxito en Buenos Aires, en la planta elaborada de quesos donde fuera aislado el fago St15, lo que alienta la posibilidad de emplear esta metodología en el desarrollo de fermentos lácticos