Potential application of phage vB_EfKS5 to control Enterococcus faecalis and its biofilm in food.

Contaminated food with antibiotic-resistant Enterococcus spp. could be the vehicle for transmitting Enterococcus to humans and accordingly cause a public health problem. The accumulation of biogenic amines produced by Enterococcus faecalis (E. faecalis) in food may have cytological effects. Bacteriophages (phage in short) are natural antimicrobial agents and can be used alone or in combination with other food preservatives to reduce food microbial contaminants. The aim of this study was to isolate a novel phage against E. faecalis and determine its host range to evaluate its potential application. Bacteriophage, vB_EfKS5, with a broad host range, was isolated to control the growth of E. faecalis. The vB_EfKS5 genome is 59,246 bp in length and has a GC content of 39.7%. The computational analysis of phage vB_EfKS5 genome confirmed that it does not contain any lysogenic, toxic, or virulent genes. Phage vB_EfKS5 exhibited lytic activity against most E. faecalis isolates with different multiplicities of infections and it infected 75.5% (22/29) of E. faecalis isolates and 42.3% (3/7) of E. faecium isolates. It was also able to destroy the biofilm formed by E. faecalis with different MOIs. Phage vB_EfKS5 alone or in combination with nisin could control the growth of E. faecalis in broth and milk. Based on its high productivity, stability, short latent period, and large burst size, phage vB_EfKS5 has a high potential for applications both in food and medical applications.

Biocontrol of Salmonella Typhimurium in milk, lettuce, raw pork meat and ready-to-eat steamed-chicken breast by using a novel bacteriophage with broad host range.

Salmonella spp., one of the most frequently reported bacteria, causes foodborne illness and economic losses. Due to the threat of increasing antibiotic resistant foodborne pathogens, application of bacteriophages as novel antibacterial agents in food matrices has become an emerging strategy. In this study, a novel Salmonella phage PS3-1 with high lytic activity against Salmonella Typhimurium was identified from previously isolated phages. PS3-1 belonged to the class Caudoviricetes with a broad host range, and had relatively short latent period (15 min), large burst size (92 PFU/cell), high pH stability (pH 3.0-11.0) and thermal tolerance (4-60 °C). Genome sequencing analysis showed that PS3-1 genome consisted of 107,110 bp DNA, without antibiotic resistance and virulence related genes. The results of growth curve and time-kill assay showed that PS3-1 not only inhibited the growth of S. Typhimurium, but also effectively decreased the viable cell counts (0.30-4.72 log) after 24-h incubation at 7, 25 and 37 °C (P < 0.05). Moreover, >1.28 log of established biofilm cells were effectively removed after 24-h treatment with PS3-1. Besides, PS3-1 significantly reduced the viability of S. Typhimurium in milk, lettuce, raw pork meat and ready-to-eat steamed-chicken breast at different temperatures (P < 0.05). These results demonstrated that PS3-1 may be an excellent antibacterial agent for controlling S. Typhimurium in food industry.

Characterization and optimization of bacteriophage cocktails to control Clostridium perfringens in vitro and in curry roux.

Clostridium perfringens is a major cause of foodborne disease in developed countries. The aim of this study was to isolate and characterize phages specific to C. perfringens to evaluate the most efficient phage cocktail for the biocontrol of C. perfringens, both in vitro and in curry roux. In this study, four phages were isolated from chicken meat and were morphologically and genetically characterized along with two phages previously isolated in our laboratory that display different host lysis spectra. Phage cocktail CP11, consisting of phages CPQ3, 7, 8, and 10, showed the broadest host range. Electron micrograph images suggested that all four phages belong to the Podoviridae family, and none of them carry any antibiotic resistance or toxin genes. Notably, the phages were stable at various pH values and in curry roux. Cocktails consisting of six, five, and four phages at the same concentrations were examined to determine the most effective phage cocktail. Phage cocktail PC11 significantly decreased the viable count of C. perfringens to a value less than the lower detection limit up to 48 h at both 8 and 37 °C in broth and at 24 °C in the curry roux. These results suggest that phage cocktail PC11 is a promising natural biocontrol agent against C. perfringens in vitro and in curry roux.

Characterization of Clostridium perfringens bacteriophages and their application in chicken meat and milk.

Clostridium perfringens is one of the most important foodborne pathogens in developed countries. It causes severe food poisoning outbreaks worldwide, along with mortality and economic losses. Recently, bacteriophages have been investigated as an alternative tool to control pathogenic bacteria in the food industry. In this study, 19 Clostridium perfringens and 6 Clostridium perfringens bacteriophages were isolated from chicken meat. According to host range and stability tests, bacteriophage CPQ1 showed high thermostability and the broadest host range. The electron micrograph image of this bacteriophage suggested that it belongs to the Picovirinae subfamily of the Podoviridae family. Nucleotide sequence analysis of the genomic DNA indicated the absence of any antibiotic resistance, toxin, or virulence genes. In broth, CPQ1 showed strong lytic activity with a low MOI of 1, decreasing the OD600 of Clostridium perfringens cell suspension from 0.2 to 0.02 at 37 °C in 2 h. In pasteurized milk and chicken meat, CPQ1 with an MOI of 10 also caused a significant decrease in viable counts of Clostridium perfringens compared to the bacteriophageless control at both 24 °C and 37 °C. This is the first report on the application of bacteriophage to control Clostridium perfringens in foods.

Serotypes, Pathogenic Potential, and Antimicrobial Resistance of Escherichia coli Isolated from Subclinical Bovine Mastitis Milk Samples in Egypt.

Subclinical mastitis (SCM) is regarded as both a problem for dairy producers and a threat to human health worldwide owing to the potential bacterial contamination of milk and dairy products, particularly those made from raw milk. In the present study, we isolated Escherichia coli from 14 (9.3%) SCM milk samples. We serotyped each E. coli isolate (n = 14), and investigated its potential pathogenicity and antimicrobial resistance (AMR). The serotyping results showed that the E. coli isolates belonged to serotypes O55:H7 (n = 2), O111:H4 (n = 2), O127:H6 (n = 2), O128:HUT (n = 2), O26:HUT (n = 1), O44:H18 (n = 1), O114:H21 (n = 1), O86:HUT (n = 1), O124:HUT (n = 1), and O127:H7 (n = 1). Potential pathogenicity was detected in 93% (13/14) of the isolates. In particular, 13 isolates possessed at least one of the examined virulence genes. Ten isolates (71%) exhibited AMR to at least one of the tested antimicrobials, four (40%) were multidrug-resistant, and one isolate produced extended-spectrum ß-lactamases. The obtained results indicate that SCM acts as a source for the spread of potentially pathogenic E. coli strains that are resistant to many groups of antimicrobials, and may constitute a hazard to both public and animal health.