Bacteriophages Isolation and Efficacy Testing.

Bacteriophages (phages) are viruses that infect bacteria and are the most abundant biological entity on the planet. Phages have gained popularity as an alternative to antibiotics due to their specificity and ability to efficiently lyse antimicrobial resistant bacterial pathogens. Before using phages, they must be isolated from the environment and tested to ensure purity and lytic ability against various hosts. This protocol walks through the entire multi-day procedure of enriching and processing raw environmental samples (seawater, primary sludge, and soil), testing for lytic activity, selecting and picking potential phage plaques, verifying phage purity, and finally, propagation (liquid and solid) of phages to obtain high-titer crude phage lysates.

Bacteriocinogenic probiotics as an integrated alternative to antibiotics in chicken production - why and how?

The misuse of antibiotics in the livestock industry has played an important role in the spread of resistant superbugs with severe health implications for humans. With the recent ban on the use of antibiotics in poultry and poultry feed in Canada and the USA, poultry farmers will have to rely on the use of alternatives to antibiotics (such as feed acidifiers, antibodies, bacteriophages, antimicrobial peptides, prebiotics, and probiotics) to maintain the same productivity and health of their livestock. Of particular interest are bacteriocinogenic probiotics, that is, bacterial strains capable of producing bacteriocins that confer health benefits on the host. These bacterial strains have multiple promising features, such as the ability to attach to the host mucosa, colonize, proliferate, and produce advantageous products such as bacteriocins and short-chain fatty acids. These not only affect pathogenic colonization but improve poultry phenotype as well. Bacteriocins are antimicrobial peptides with multiple promising features such as being non-harmful for human and animal consumption, non-disruptive to the host microbiota eubiosis, non-cytotoxic, and non-carcinogenic. Therefore, bacteriocinogenic probiotics are at the forefront to be excellent candidates for effective replacements to antibiotics. While evidence of their safety and effectiveness is accumulating in vitro and in vivo in inhibiting pathogens while promoting animal health, their safety and history of use in livestock remains unclear and requires additional investigations. In the present paper, we review the safety assessment regulations and commercialization policies on existing and novel bacteriocinogenic and bacteriocin products intended to be used in poultry feed as an alternative to antibiotics.

Dual Inhibition of Salmonella enterica and Clostridium perfringens by New Probiotic Candidates Isolated from Chicken Intestinal Mucosa.

The poultry industry is the fastest-growing agricultural sector globally. With poultry meat being economical and in high demand, the end product's safety is of importance. Globally, governments are coming together to ban the use of antibiotics as prophylaxis and for growth promotion in poultry. Salmonella and Clostridium perfringens are two leading pathogens that cause foodborne illnesses and are linked explicitly to poultry products. Furthermore, numerous outbreaks occur every year. A substitute for antibiotics is required by the industry to maintain the same productivity level and, hence, profits. We aimed to isolate and identify potential probiotic strains from the ceca mucosa of the chicken intestinal tract with bacteriocinogenic properties. We were able to isolate multiple and diverse strains, including a new uncultured bacterium, with inhibitory activity against Salmonella Typhimurium ATCC 14028, Salmonella Abony NCTC 6017, Salmonella Choleraesuis ATCC 10708, Clostridium perfringens ATCC 13124, and Escherichia coli ATCC 25922. The five most potent strains were further characterized for their probiotic potential (i.e., sensitivity to antibiotics and tolerance to gastrointestinal physicochemical conditions). Our analyzed lactobacilli strains exhibited some interesting probiotic features while being inhibitory against targeted pathogens.

Development of prototypes of bioactive packaging materials based on immobilized bacteriophages for control of growth of bacterial pathogens in foods.

Due to lack of adequate control methods to prevent contamination in fresh produce and growing consumer demand for natural products, the use of bacteriophages has emerged as a promising approach to enhance safety of these foods. This study sought to control Listeria monocytogenes in cantaloupes and RTE meat and Escherichia coli O104:H4 in alfalfa seeds and sprouts under different storage conditions by using specific lytic bacteriophage cocktails applied either free or immobilized. Bacteriophage cocktails were introduced into prototypes of packaging materials using different techniques: i) immobilizing on positively charged modified cellulose membranes, ii) impregnating paper with bacteriophage suspension, and iii) encapsulating in alginate beads followed by application of beads onto the paper. Phage-treated and non-treated samples were stored for various times and at temperatures of 4°C, 12°C or 25°C. In cantaloupe, when free phage cocktail was added, L. monocytogenes counts dropped below the detection limit of the plating technique (<1 log CFU/g) after 5 days of storage at both 4°C and 12°C. However, at 25°C, counts below the detection limit were observed after 3 and 6h and a 2-log CFU/g reduction in cell numbers was seen after 24h. For the immobilized Listeria phage cocktail, around 1-log CFU/g reduction in the Listeria count was observed by the end of the storage period for all tested storage temperatures. For the alfalfa seeds and sprouts, regardless of the type of phage application technique (spraying of free phage suspension, bringing in contact with bacteriophage-based materials (paper coated with encapsulated bacteriophage or impregnated with bacteriophage suspension)), the count of E. coli O104:H4 was below the detection limit (<1 log CFU/g) after 1h in seeds and about a 1-log cycle reduction in E. coli count was observed on the germinated sprouts by day 5. In ready-to-eat (RTE) meat, LISTEX™ P100, a commercial phage product, was able to significantly reduce the growth of L. monocytogenes at both storage temperatures, 4°C and 10°C, for 25 days regardless of bacteriophage application format (immobilized or non-immobilized (free)). In conclusion, the developed phage-based materials demonstrated significant antimicrobial effect, when applied to the artificially contaminated foods, and can be used as prototypes for developing bioactive antimicrobial packaging materials capable of enhancing the safety of fresh produce and RTE meat.