Bacteriophages safely reduce Salmonella contamination in pet food and raw pet food ingredients.

Contamination of pet food with Salmonella is a serious public health concern, and several disease outbreaks have recently occurred due to human exposure to Salmonella tainted pet food. The problem is especially challenging for raw pet foods (which include raw meats, seafood, fruits, and vegetables). These foods are becoming increasingly popular because of their nutritional qualities, but they are also more difficult to maintain Salmonella-free because they lack heat-treatment. Among various methods examined to improve the safety of pet foods (including raw pet food), one intriguing approach is to use bacteriophages to specifically kill Salmonella serotypes. At least 2 phage preparations (SalmoFresh® and Salmonelex™) targeting Salmonella are already FDA cleared for commercial applications to improve the safety of human foods. However, similar preparations are not yet available for pet food applications. Here, we report the results of evaluating one such preparation (SalmoLyse®) in reducing Salmonella levels in various raw pet food ingredients (chicken, tuna, turkey, cantaloupe, and lettuce). Application of SalmoLyse® in low (ca. 2-4×106 PFU/g) and standard (ca. 9×106 PFU/g) concentrations significantly (P < 0.01) reduced (by 60-92%) Salmonella contamination in all raw foods examined compared to control treatments. When SalmoLyse®-treated (ca. 2×107 PFU/g) dry pet food was fed to cats and dogs, it did not trigger any deleterious side effects in the pets. Our data suggest that the bacteriophage cocktail lytic for Salmonella can significantly and safely reduce Salmonella contamination in various raw pet food ingredients.

Bacteriophage cocktail for biocontrol of Salmonella in dried pet food.

Human salmonellosis has been associated with contaminated pet foods and treats. Therefore, there is interest in identifying novel approaches for reducing the risk of Salmonella contamination within pet food manufacturing environments. The use of lytic bacteriophages shows promise as a safe and effective way to mitigate Salmonella contamination in various food products. Bacteriophages are safe, natural, highly targeted antibacterial agents that specifically kill bacteria and can be targeted to kill food pathogens without affecting other microbiota. In this study, we show that a cocktail containing six bacteriophages had a broadspectrum activity in vitro against a library of 930 Salmonella enterica strains representing 44 known serovars. The cocktail was effective against 95% of the strains in this tested library. In liquid culture dose-ranging experiments, bacteriophage cocktail concentrations of ≥10(8) PFU/ml inactivated more than 90% of the Salmonella population (10(1) to 10(3) CFU/ml). Dried pet food inoculated with a mixture containing equal proportions of Salmonella serovars Enteritidis (ATCC 4931), Montevideo (ATCC 8387), Senftenberg (ATCC 8400), and Typhimurium (ATCC 13311) and then surface treated with the six-bacteriophage cocktail (≥2.5 ± 1.5 × 10(6) PFU/g) achieved a greater than 1-log (P < 0.001) reduction compared with the phosphate-buffered saline-treated control in measured viable Salmonella within 60 min. Moreover, this bacteriophage cocktail reduced natural contamination in samples taken from an undistributed lot of commercial dried dog food that tested positive for Salmonella. Our results indicate that bacteriophage biocontrol of S. enterica in dried pet food is technically feasible.

Tetrahymena Pot2 is a developmentally regulated paralog of Pot1 that localizes to chromosome breakage sites but not to telomeres.

Tetrahymena telomeres are protected by a protein complex composed of Pot1, Tpt1, Pat1, and Pat2. Pot1 binds the 3' overhang and serves multiple roles in telomere maintenance. Here we describe Pot2, a paralog of Pot1 which has evolved a novel function during Tetrahymena sexual reproduction. Pot2 is unnecessary for telomere maintenance during vegetative growth, as the telomere structure is unaffected by POT2 macronuclear gene disruption. Pot2 is expressed only in mated cells, where it accumulates in developing macronuclei around the time of two chromosome processing events: internal eliminated sequence (IES) excision and chromosome breakage. Chromatin immunoprecipitation (ChIP) demonstrated Pot2 localization to regions of chromosome breakage but not to telomeres or IESs. Pot2 association with chromosome breakage sites (CBSs) occurs slightly before chromosome breakage. Pot2 did not bind CBSs or telomeric DNA in vitro, suggesting that it is recruited to CBSs by another factor. The telomere proteins Pot1, Pat1, and Tpt1 and the IES binding factor Pdd1 fail to colocalize with Pot2. Thus, Pot2 is the first protein found to associate specifically with CBSs. The selective association of Pot2 versus Pdd1 with CBSs or IESs indicates a mechanistic difference between the chromosome processing events at these two sites. Moreover, ChIP revealed that histone marks characteristic of IES processing, H3K9me3 and H3K27me3, are absent from CBSs. Thus, the mechanisms of chromosome breakage and IES excision must be fundamentally different. Our results lead to a model where Pot2 directs chromosome breakage by recruiting telomerase and/or the endonuclease responsible for DNA cleavage to CBSs.

Bacteriophages lytic for Salmonella rapidly reduce Salmonella contamination on glass and stainless steel surfaces.

A cocktail of six lytic bacteriophages, SalmoFresh™, significantly (p < 0.05) reduced the number of surface-applied Salmonella Kentucky and Brandenburg from stainless steel and glass surfaces by > 99% (2.1-4.3 log). Both strains were susceptible to SalmoFresh™ in the spot-test assay. Conversely, SalmoFresh™ was unable to reduce surface contamination with a Salmonella Paratyphi B strain that was not susceptible to the phage cocktail in the spot-test assay. However, by replacing two SalmoFresh™ component phages with two new phages capable of lysing the Paratyphi B strain in the spot-test assay, the target range of the cocktail was shifted to include the Salmonella Paratyphi B strain. The modified cocktail, SalmoLyse™, was able to significantly (p < 0.05) reduce surface contamination of the Paratyphi B strain by > 99% (2.1-4.1 log). The data show that both phage cocktails were effective in significantly reducing the levels of Salmonella on hard surfaces, provided the contaminating strains were susceptible in the spot-test (i.e., spot-test susceptibility was indicative of efficacy in subsequent surface decontamination studies). The data also support the concept that phage preparations can be customized to meet the desired antibacterial application.