RESUMEN
AIMS: To assay the combination of phage and probiotics against EHEC in vitro on infected Hep-2 cells. METHODS AND RESULTS: Phage and probiotics treatments on EHEC O157:H7-infected Hep-2 cells were assayed individually or combined. The effect of freeze-drying on phage and probiotic antimicrobial activity was also studied. While treatment with phage alone increased cell detachment caused by EHEC infection, the treatments with MM alone or in combination with phage proved to effectively diminish cell damage caused by EHEC infection. Combined treatment showed a decrease in apoptotic cell count of 57·3% and a reduction in EHEC adhesion to cell monolayer of 1·2 log CFU. The simultaneous use of phage and probiotics showed no antagonistic effect, and freeze-drying did not affect their antipathogenic activity. CONCLUSIONS: The combination of phage and probiotics has great potential for reducing the number of pathogens adhered to epithelial cells during EHEC O157:H7 infection and attenuating the cytotoxic effect derived from it. Further in vivo assays are needed for assessing the actual effectiveness of the treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a freeze-dried formulation of phage and probiotics capable of controlling EHEC infections and reducing epithelial cell damage in vitro.
Asunto(s)
Bacteriófagos/patogenicidad , Infecciones por Escherichia coli/tratamiento farmacológico , Escherichia coli O157/efectos de los fármacos , Probióticos/farmacología , Adhesión Bacteriana/efectos de los fármacos , Células Epiteliales/microbiología , Infecciones por Escherichia coli/metabolismo , Escherichia coli O157/virología , Humanos , Probióticos/uso terapéuticoRESUMEN
The aims of this study were to determine the stability of Podoviridae coliphage CA933P during lyophilization and storage in different media, and to establish similarities between the results obtained and those expected through mechanisms described for proteins stabilization during freeze-drying. PBS and SM buffer were assayed as lyophilization media. The effect of inorganic salts concentration as well as the addition of disaccharides on phage stability during freeze-drying and storage was also studied. The addition of low sucrose concentration (0.1 mol l⻹) to SM buffer stabilized phage during freezing and drying steps of the lyophilization process, but higher sugar concentrations were detrimental to phage stability during freeze-drying. Sucrose stabilized phage during storage for at least 120 days. The lyoprotective effect of low concentrations of disaccharides during the drying step of the lyophilization of proteins as well as the stabilization of the freeze-dried product in time correlated with the results obtained for phage CA933P.
Asunto(s)
Crioprotectores/metabolismo , Liofilización/métodos , Podoviridae/fisiología , Sacarosa/metabolismo , Tampones (Química)RESUMEN
AIMS: To isolate, characterize and select phages as potential biocontrol agents of enterohemorrhagic and Shiga toxin-producing Escherichia coli (EHEC and STEC) in cattle. METHODS AND RESULTS: Sixteen STEC and EHEC coliphages were isolated from bovine minced meat and stool samples and characterized with respect to their host range against STEC, EHEC and other Gram-negative pathogens; their morphology by electron microscopy; the presence of the stx1, stx2 and cI genes by means of PCR; RAPD and rep-PCR profiles; plaque formation; and acid resistance. Six isolates belonged to the Myoviridae and 10 to the Podoviridae families. The phages negative for stx and cI that formed large, well-defined plaques were all isolated using EHEC O157:H7 as host. Among them, only CA911 was a myophage and, together with CA933P, had the broadest host range for STEC and EHEC; the latter phage also infected Shigella and Pseudomonas. Isolates CA911, MFA933P and MFA45D differed in particle morphology and amplification patterns by RAPD and rep-PCR and showed the highest acidity tolerance. CONCLUSIONS: Myophage CA911 and podophages CA933P, MFA933P and MFA45D were chosen as the best candidates for biocontrol of STEC and EHEC in cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: This work employs steps for a rational selection and characterization of bacteriophages as therapeutic agents. This report constitutes the first documentation of STEC and EHEC phages isolated in Argentina and proposes for the first time the use of rep-PCR as a complement of RAPD on DNA fingerprinting of phages.
Asunto(s)
Colifagos/aislamiento & purificación , Escherichia coli Enterohemorrágica/virología , Escherichia coli Shiga-Toxigénica/virología , Animales , Argentina , Bovinos , Colifagos/genética , Colifagos/ultraestructura , Heces/virología , Genes Virales , Especificidad del Huésped , Carne/virología , Reacción en Cadena de la Polimerasa , Factores de Virulencia/genéticaRESUMEN
DNA fingerprints of lactic acid bacteria were generated by polymerase chain reaction using a primer based on the repetitive elements found in the genome of Streptococcus pneumoniae (BOX-PCR). The method made it possible to identify 37 isolates from raw milk. industrial starters and yogurt. Differentiation at species, subspecies and strain level was possible for Lactobacillus delbrueckii subsp. lactis, Lb. delbrueckii subsp bulgaricus and Str. thermophilus. BOX-PCR was also applied to studying the strain composition of a starter culture and the direct detection of strains in commercial fermented milk.