Denis A. Spricigo

Use of a bacteriophage cocktail to control Salmonella in food and the food industry.

The use of lytic bacteriophages for the biocontrol of food-borne pathogens in food and in the food industry is gaining increasing acceptance. In this study, the effectiveness of a bacteriophage cocktail composed of three different lytic bacteriophages (UAB_Phi 20, UAB_Phi78, and UAB_Phi87) was determined in four different food matrices (pig skin, chicken breasts, fresh eggs, and packaged lettuce) experimentally contaminated with Salmonella enterica serovar Typhimurium and S. enterica serovar Enteritidis. A significant bacterial reduction (>4 and 2 log/cm(2) for S. Typhimurium and S. Enteritidis, respectively; p≤0.005) was obtained in pig skin sprayed with the bacteriophage cocktail and then incubated at 33 °C for 6h. Significant decreases in the concentration of S. Typhimurium and S. Enteritidis were also measured in chicken breasts dipped for 5 min in a solution containing the bacteriophage cocktail and then refrigerated at 4 °C for 7 days (2.2 and 0.9 log10 cfu/g, respectively; p≤0.0001) as well as in lettuce similarly treated for 60 min at room temperature (3.9 and 2.2 log10 cfu/g, respectively; p≤0.005). However, only a minor reduction of the bacterial concentration (0.9 log10 cfu/cm(2) of S. Enteritidis and S. Typhimurium; p≤0.005) was achieved in fresh eggs sprayed with the bacteriophage cocktail and then incubated at 25 °C for 2 h. These results show the potential effectiveness of this bacteriophage cocktail as a biocontrol agent of Salmonella in several food matrices under conditions similar to those used in their production.

Significance of the Bacteriophage Treatment Schedule in Reducing Salmonella Colonization of Poultry

ABSTRACT Salmonella remains the major cause of food-borne diseases worldwide, with chickens known to be the main reservoir for this zoonotic pathogen. Among the many approaches to reducing Salmonella colonization of broilers, bacteriophage offers several advantages. In this study, three bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) obtained from our collection that exhibited a broad host range against Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium were characterized with respect to morphology, genome size, and restriction patterns. A cocktail composed of the three bacteriophages was more effective in promoting the lysis of S. Enteritidis and S. Typhimurium cultures than any of the three bacteriophages alone. In addition, the cocktail was able to lyse the Salmonella enterica serovars Virchow, Hadar, and Infantis. The effectiveness of the bacteriophage cocktail in reducing the concentration of S. Typhimurium was tested in two animal models using different treatment schedules. In the mouse model, 50% survival was obtained when the cocktail was administered simultaneously with bacterial infection and again at 6, 24, and 30 h postinfection. Likewise, in the White Leghorn chicken specific-pathogen-free (SPF) model, the best results, defined as a reduction of Salmonella concentration in the chicken cecum, were obtained when the bacteriophage cocktail was administered 1 day before or just after bacterial infection and then again on different days postinfection. Our results show that frequent treatment of the chickens with bacteriophage, and especially prior to colonization of the intestinal tract by Salmonella, is required to achieve effective bacterial reduction over time.

Phagomagnetic separation and electrochemical magneto-genosensing of pathogenic bacteria.

This paper addresses the use of bacteriophages immobilized on magnetic particles for the biorecognition of the pathogenic bacteria, followed by electrochemical magneto-genosensing of the bacteria. The P22 bacteriophage specific to Salmonella (serotypes A, B, and D1) is used as a model. The bacteria are captured and preconcentrated by the bacteriophage-modified magnetic particles through the host interaction with high specificity and efficiency. DNA amplification of the captured bacteria is then performed by double-tagging polymerase chain reaction (PCR). Further detection of the double-tagged amplicon is achieved by electrochemical magneto-genosensing. The strategy is able to detect in 4 h as low as 3 CFU mL(-1) of Salmonella in Luria-Bertani (LB) media. This approach is compared with conventional culture methods and PCR-based assay, as well as with immunological screening assays for bacteria detection, highlighting the outstanding stability and cost-efficient and animal-free production of bacteriophages as biorecognition element in biosensing devices.

Genomics of Three New Bacteriophages Useful in the Biocontrol of Salmonella.

Non-typhoid Salmonella is the principal pathogen related to food-borne diseases throughout the world. Widespread antibiotic resistance has adversely affected human health and has encouraged the search for alternative antimicrobial agents. The advances in bacteriophage therapy highlight their use in controlling a broad spectrum of food-borne pathogens. One requirement for the use of bacteriophages as antibacterials is the characterization of their genomes. In this work, complete genome sequencing and molecular analyses were carried out for three new virulent Salmonella-specific bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) able to infect a broad range of Salmonella strains. Sequence analysis of the genomes of UAB_Phi20, UAB_Phi78, and UAB_Phi87 bacteriophages did not evidence the presence of known virulence-associated and antibiotic resistance genes, and potential immunoreactive food allergens. The UAB_Phi20 genome comprised 41,809 base pairs with 80 open reading frames (ORFs); 24 of them with assigned function. Genome sequence showed a high homology of UAB_Phi20 with Salmonella bacteriophage P22 and other P22likeviruses genus of the Podoviridae family, including ST64T and ST104. The DNA of UAB_Phi78 contained 44,110 bp including direct terminal repeats (DTR) of 179 bp and 58 putative ORFs were predicted and 20 were assigned function. This bacteriophage was assigned to the SP6likeviruses genus of the Podoviridae family based on its high similarity not only with SP6 but also with the K1-5, K1E, and K1F bacteriophages, all of which infect Escherichia coli. The UAB_Phi87 genome sequence consisted of 87,669 bp with terminal direct repeats of 608 bp; although 148 ORFs were identified, putative functions could be assigned to only 29 of them. Sequence comparisons revealed the mosaic structure of UAB_Phi87 and its high similarity with bacteriophages Felix O1 and wV8 of E. coli with respect to genetic content and functional organization. Phylogenetic analysis of large terminase subunits confirms their packaging strategies and grouping to the different phage genus type. All these studies are necessary for the development and the use of an efficient cocktail with commercial applications in bacteriophage therapy against Salmonella.

Remarkable diversity of Salmonella bacteriophages in swine and poultry

The diversity of 55 Salmonella-specific bacteriophages isolated from 191 fecal samples of poultry and swine from farms located in diverse geographic areas of Spain was determined using lysis profiling, DNA restriction and random amplification of polymorphic DNA (RAPD-PCR). Among them, lysis profiling and RAPD-PCR exhibited 100% typeability and DNA restriction 96%, with discriminatory power of 0.978 (± 0.016), 0.938 (± 0.028) and 0.982 (± 0.013), respectively. The highest concordance (0.974) was that between RAPD-PCR and lysis profiling. None of the bacteriophages isolated from poultry and swine shared any DNA restriction or RAPD-PCR patterns and only two lysis profiles were common to bacteriophages isolated from poultry and swine. The major part of the lysis and RAPD-PCR profiles from the bacteriophages isolated from poultry included only one or two bacteriophages, while those obtained from swine contained more than two bacteriophages. Overall, our results provide evidence of the remarkable diversity exhibited by bacteriophages of Salmonella in farm animals. Moreover, they also show that RAPD-PCR may also be suitable for the pre-screening of the diversity of Salmonella bacteriophages for further use in biocontrol and therapeutic strategies.

Relevance of DNA Alkylation Damage Repair Systems in Salmonella enterica Virulence

Systematic inactivation of pathways involved in DNA alkylation damage repair demonstrated that inactivation of the ada, ogt, tag, uvrA, and mfd genes is required to detect a Salmonella enterica virulence decrease. Furthermore, the fitness of S. enterica, defective in these genes, is lowered only when the bacterium is orally, but not intraperitoneally, inoculated.

Significance of tagI and mfd genes in the virulence of non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae (NTHi) is an opportunist pathogen well adapted to the human upper respiratory tract and responsible for many respiratory diseases. In the human airway, NTHi is exposed to pollutants, such as alkylating agents, that damage its DNA. In this study, we examined the significance of genes involved in the repair of DNA alkylation damage in NTHi virulence. Two knockout mutants, tagI and mfd, encoding N³-methyladenine-DNA glycosylase I and the key protein involved in transcription-coupled repair, respectively, were constructed and their virulence in a BALB/c mice model was examined. This work shows that N³-methyladenine-DNA glycosylase I is constitutively expressed in NTHi and that it is relevant for its virulence.