Isolation and Characterization of Bacteriophage ZCSE6 against Salmonella spp.: Phage Application in Milk

Abstract

Food safety is very important in the food industry as most pathogenic bacteria can cause food-borne diseases and negatively affect public health. In the milk industry, contamination with Salmonella has always been a challenge, but the risks have dramatically increased as almost all bacteria now show resistance to a wide range of commercial antibiotics. This study aimed to isolate a bacteriophage to be used as a bactericidal agent against Salmonella in milk and dairy products. Here, phage ZCSE6 has been isolated from raw milk sample sand molecularly and chemically characterized. At different multiplicities of infection (MOIs) of 0.1, 0.01, and 0.001, the phage–Salmonella interaction was studied for 6 h at 37 °C and 24 h at 8 °C. In addition, ZCSE6 was tested against Salmonella contamination in milk to examine its lytic activity for 3 h at 37 °C. The results showed that ZCSE6 has a small genome size (<48.5 kbp) and belongs to the Siphovirus family. Phage ZCSE6 revealed a high thermal and pH stability at various conditions that mimic milk manufacturing and supply chain conditions. It also demonstrated a significant reduction in Salmonella concentration in media at various MOIs, with higher bacterial eradication at higher MOI. Moreover, it significantly reduced Salmonella growth (MOI 1) in milk, manifesting a 1000-fold decrease in bacteria concentration following 3 h incubation at 37 °C. The results highlighted the strong ability of ZCSE6 to kill Salmonella and control its growth in milk. Thus, ZCSE6 is recommended as a biocontrol agent in milk to limit bacterial growth and increase the milk shelf-life.

Isolation, characterization, and application of a novel polyvalent lytic phage STWB21 against typhoidal and nontyphoidal Salmonella spp.

“…In this study, we have characterized a lytic bacteriophage STWB21 isolated from the lake water sample in the outskirts of Kolkata area using a strain of S. typhi as the host bacterium. Phage STWB21 was found to be effective against many other strains of typhoidal and non-typhoidal Salmonella which indicates the high specificity of STWB21 against Salmonella ( Abdelsattar et al, 2021 ). On the other hand, the ability of phage STWB21 to lyse Sh.…”

Evaluation of bacteriophage to reduce Escherichia coli contamination in different food samples

“…The phage ZCECO 5 was centrifuged for half an hour at 12,000 rpm and resuspended in 100 µL SM buffer at a 10 10 PFU/ml titer [54]. Morphology was examined on glow discharged (1 minute under vacuum) by TEM at the Faculty of Science, Alexandria University, Egypt [44], [50], [66]. The phage solution was placed on Formvar/carbon-coated copper grid (Pelco International) and fixated with glutaraldehyde (2.5 per cent v/v), rinsed, and dyed with 2 per cent phosphotungstic acid.…”

“…The genome size of ZCECO 5 phage (10 10 PFU/mL) DNA was estimated using pulsed-field gel electrophoresis [50], [67]. First, plugs of phage were made as previously reported, [67] by putting 200 µL of phage in a 200 µL agarose gel made from TE buffer 1x (Tris-HCl, Loba Chemie, India, EDTA, Fisher chemical, Pittsburgh, PA, USA; pH 8) and 1.4 per cent agarose (Lonza, Switzerland), and drying for 15 minutes [66]. The phage was then placed into agarose plugs and digested by lysis buffer (1 per cent w/v NLauryl sarcosine [Sigma Aldrich, Gillingham, UK]; 0.2 per cent w/v SDS [Sigma Aldrich, Gillingham, UK]; 100 mM EDTA; 1 mg/ml Proteinase K [ThermoFischer Scientific], Waltham, MA, USA; pH 8), for 18 hours at 55 °C with slight shaking for lysis of the phage capsid and digesting protein parts [50].…”

“…After being rinsed by washing buffer, two slices (2 mm) of agarose possessing DNA have been put into the wells containing 1 per cent w/v agarose gel [50,54]. The gel had been prepared with Tris-borate-EDTA buffer 0.5x (TBE buffer) (Tris-HCl, boric acid, Fisher chemical, USA, EDTA; pH8), and the run was done with 2700 ml of 0.5x TBE buffer [66]. Using a Bio-Rad CHEF DRII system, the phage plugs were placed into the gel and then ran for 18 hours at 14 °C at 200 Volts (6 V/cm) with a switching time of 3s start to 15s end [50,54,66].…”

Evaluation of phage—antibiotic combinations in the treatment of extended-spectrum β-lactamase-producing Salmonella enteritidis strain PT1

“…Several research findings indicated that different types of Salmonella bacteriophages have shown promising results against various Salmonella strains [ 23 , 24 ]. According to Abdelsattar et al [ 25 ], a lytic phage, ZCSE6, reduced Salmonella growth (MOI 1) by 1000-fold in milk following 3 h of incubation at 37 °C. In a different study, two lytic phages, SPHG1 and SPHG3, and their cocktail resulted in a significant reduction in the viable count of S. Typhimurium in milk, water, and on chicken breast (at MOIs of 100 or 1000).…”