A Bacteriophage Cocktail Eliminates Salmonella Typhimurium from the Human Colonic Microbiome while Preserving Cytokine Signaling and Preventing Attachment to and Invasion of Human Cells by Salmonella In Vitro.

Nontyphoidal Salmonella strains continue to be a major cause of foodborne illness globally. One intriguing approach to reducing the risk of salmonellosis is the direct ingestion of phages targeting Salmonella to enhance natural gut resilience and provide protection during foodborne disease outbreaks. We evaluated the ability of a prophylactically administered bacteriophage cocktail, the foodborne outbreak pill (FOP) targeting Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella, to resolve a Salmonella infection in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME), a simulated gut platform populated by the human intestinal microbiome of healthy donors. The FOP preparation eliminated Salmonella enterica serovar Typhimurium from the colon compartment of the SHIME platform but health-associated metabolites, such as short-chain fatty acids and lactate, remained stable or increased in a donor-dependent manner. In studies of human intestinal cells, pretreatment of Salmonella Typhimurium with the FOP cocktail preserved lipopolysaccharide-stimulated signaling in a Caco-2-THP-1 Transwell system and prevented destruction of the Caco-2 monolayer by Salmonella. Adhesion and invasion of intestinal epithelial cells by Salmonella-a critical factor in Salmonella pathogenesis-was blunted when the bacteria were incubated with the FOP preparation before addition to the monolayer. The FOP phage cocktail was effective for (i) eliminating Salmonella from a simulated human gut without disturbing the indigenous microbiota and (ii) reducing the risk of invasion by Salmonella into the intestinal epithelia. These results suggest that the FOP preparation may be of value for reducing the risk of salmonellosis in humans, e.g., during foodborne disease outbreaks.

Isolation, characterization and efficacy of phage MJ2 against biofilm forming multi-drug resistant Enterobacter cloacae.

Biofilm is involved in a variety of infections, playing a critical role in the chronicity of infections. Enterobacter cloacae is a biofilm-forming and multi-drug-resistant (MDR) nosocomial pathogen leading to significant morbidity and mortality. This study aimed at isolation of a bacteriophage against MDR clinical strain of E. cloacae and its efficacy against bacterial planktonic cells and biofilm. A bacteriophage MJ2 was successfully isolated from wastewater and was characterized. The phage exhibited a wide range of thermal and pH stability and demonstrated considerable adsorption to host bacteria in the presence of CaCl2 or MgCl2. Transmission electron microscopy (TEM) showed MJ2 head as approximately 62 and 54 nm width and length, respectively. It had a short non-contractile tail and was characterized as a member of the family Podoviridae [order Caudovirales]. The phage MJ2 was found to possess 11 structural proteins (12-150 kDa) and a double-stranded DNA genome with an approximate size of 40 kb. The log-phase growth of E. cloacae both in biofilm and suspension was significantly reduced by the phage. The E. cloacae biofilm was formed under different conditions to evaluate the efficacy of MJ2 phage. Variable reduction pattern of E. cloacae biofilm was observed while treating it for 4 h with MJ2, i.e., biofilm under static conditions. The renewed media with intervals of 24, 72, and 120 h showed biomass decline of 2.8-, 3-, and 3.5-log, respectively. Whereas, the bacterial biofilm formed with dynamic conditions with refreshing media after 24, 72, and 120 h demonstrated decline in growth at 2.5-, 2.6-, and 3.3-log, respectively. It was, therefore, concluded that phage MJ2 possessed considerable inhibitory effects on MDR E. cloacae both in planktonic and biofilm forms.

Isolation and characterization of a bacteriophage and its utilization against multi-drug resistant Pseudomonas aeruginosa-2995.

AIMS: To identify, isolate, and characterize a lytic bacteriophage against the multiple-drug resistant clinical strain of Pseudomonas aeruginosa-2995 and to determine the phage efficacy against the bacterial planktonic cells and the biofilm. MAIN METHODS: Wastewater was used to isolate a bacteriophage. The phage was characterized with Transmission electron microscopy (TEM). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used to identify the expressed proteins. Bacteria were cultured in both suspension and biofilm to check and compare their susceptibility to phage lytic action. The activity of the phage (determined as AZ1) was determined against P. aeruginosa-2995 in both planktonic cells and the biofilm. KEY FINDINGS: A bacteriophage, designated as AZ1, was isolated from waste water showing a narrow host range. AZ1 was characterized by TEM and could be identified as an isolate in the family Siphoviridae [order Caudovirals]. Seventeen structural proteins ranging from about 12 to 110kDa were found through SDS-PAGE analysis. Its genome was confirmed as dsDNA with a length of approx. 50kb. The log-phase growth of P. aeruginosa-2995 was significantly reduced after treatment with AZ1 (4.50×108 to 2.1×103CFU/ml) as compared to control. Furthermore, phage AZ1 significantly reduced 48h old biofilm biomass about 3-fold as compared to control. SIGNIFICANCE: Pseudomonas aeruginosa is a ubiquitous free-living opportunistic human pathogen characterized by high antibiotic tolerance and tendency for biofilm formation. The phage, identified in this study, AZ1, showed promising activity in the destruction of both planktonic cells and biofilm of P. aeruginosa-2995. However, complete eradication may require a combination of phages.

Bacteriophage preparation lytic for Shigella significantly reduces Shigella sonnei contamination in various foods.

ShigaShield™ is a phage preparation composed of five lytic bacteriophages that specifically target pathogenic Shigella species found in contaminated waters and foods. In this study, we examined the efficacy of various doses (9x105-9x107 PFU/g) of ShigaShield™ in removing experimentally added Shigella on deli meat, smoked salmon, pre-cooked chicken, lettuce, melon and yogurt. The highest dose (2x107 or 9x107 PFU/g) of ShigaShield™ applied to each food type resulted in at least 1 log (90%) reduction of Shigella in all the food types. There was significant (P<0.01) reduction in the Shigella levels in all phage treated foods compared to controls, except for the lowest phage dose (9x105 PFU/g) on melon where reduction was only ca. 45% (0.25 log). The genomes of each component phage in the cocktail were fully sequenced and analyzed, and they were found not to contain any "undesirable genes" including those listed in the US Code for Federal Regulations (40 CFR Ch1). Our data suggest that ShigaShield™ (and similar phage preparations with potent lytic activity against Shigella spp.) may offer a safe and effective approach for reducing the levels of Shigella in various foods that may be contaminated with the bacterium.

Characterization of Siphoviridae phage Z and studying its efficacy against multidrug-resistant Klebsiella pneumoniae planktonic cells and biofilm.

Biofilm has many serious consequences for public health and is a major virulence factor contributing to the chronicity of infections. The aim of the current study was to isolate and characterize a bacteriophage that inhibits multidrug-resistant Klebsiella pneumonia (M) in planktonic form as well as biofilm. This phage, designated bacteriophage Z, was isolated from wastewater. Its adsorption rate to its host bacterium was significantly enhanced by MgCl2 and CaCl2. It has a wide range of pH and heat stability. From its one-step growth, latent time and burst size were determined to be 24 min and about 320 virions per cell, respectively. As analysed by transmission electron microscopy, phage Z had an icosahedral head of width 76±10 nm, length 92±14 nm and icosahedron side 38 nm, and a non-contractile tail 200±15 nm long and 14-29 nm wide. It belongs to the family Siphoviridae in the order Caudovirales. Six structural proteins ranging from 18 to 65 kDa in size were revealed by SDS-PAGE. The genome was found to comprise double-stranded DNA with an approximate size of 36 kb. Bacteria were grown in suspension and as biofilms to compare the susceptibility of both phenotypes to the phage lytic action. Phage Z was effective in reducing biofilm biomass after 24 and 48 h, showing more than twofold and threefold reduction, respectively. Biofilm cells and stationary-phase planktonic bacteria were killed at a lower rate than exponential-phase planktonic bacteria.

Characterization of new Myoviridae bacteriophage WZ1 against multi-drug resistant (MDR) Shigella dysenteriae.

Shigella dysenteriae is a normal inhabitant of the human gastrointestinal tract, but sometimes it causes severe infection known as shigellosis (bacillary dysentery). Bacteriophages are considered very safe and effective agents for controlling bacterial infections and contaminations. In this study, we describe the isolation and characterization of bacteriophage WZ1, isolated from waste water which inhibits the growth of S. dysenteriae. Phage WZ1 showed maximum stability at 37 °C and was stable up to 65 °C but was totally inactive at 70 °C. The pH stability increased from low to high and was totally inactive at pH 3 while maximum stability was observed at optimal pH 7. Phage WZ1 adsorption rate to the host bacterium was significantly enhanced by the addition of CaCl2 . It has a latent time and burst time of 24 min and about 430 virions/cell, respectively. Transmission electron microscopy of phage WZ1 revealed a head width of 10 ± 0.5 nm and length of 10 ± 0.2 nm with a contractile tail of 128 ± 25 nm long and 21 ± 0.5 nm wide and belongs to family Myoviridae of order Caudovirales. Twelve structural proteins ranging from 22 to 150 kDa were detected by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The genome was found to be double stranded DNA with an approximate size of 38 kb. It has a very good reduction potential for S. dysenteriae by lowering abruptly the optical density of the planktonic S. dysenteriae culture. Phage WZ1 is a very promising candidate for phage therapy and other applications such as phage typing.

Isolation and Characterization of a Myoviridae MJ1 Bacteriophage Against Multi-Drug Resistant Escherichia coli 3.

BACKGROUND: Antibiotic resistance in Escherichia coli, a member of the Enterobacteriaceae, is of particular concern because it is the most common (Gram-negative) pathogen causing nosocomial and community infections. Researchers are now considering the use of phages for the control of various antibiotic-resistant bacterial infections. OBJECTIVES: The purpose of this study was to isolate and characterize a novel pathogenic/lytic phage that targets multi-drug resistant (MDR) E. coli 3, and to investigate its effectiveness at lysing this bacterium. MATERIALS AND METHODS: A clinical strain of E. coli 3 was identified based on its 16S rRNA sequencing and its antibiotic resistance profile was determined by the disc diffusion method. A bacteriophage was isolated from wastewater and its various characteristics, such as host range, heat tolerance, pH stability, one step growth, total protein content, and genome size, were determined. The antibacterial property of the phage was determined against log-phase bacterial planktonic cells at 37°C. RESULTS: The bacteriophage, designated MJ1, was isolated by testing against a clinical MDR E. coli 3 strain. The MJ1 phage showed a wide range of heat and pH stability. The phage morphology, determined by transmission electron microscopy, revealed a structure comprised of a head (108 ± 0.2 nm long by 128 ± 0.5 nm wide) and a contractile tail (123 ± 0.5 nm long by 15 - 26 nm wide). These features placed the MJ1 phage in the family Myoviridae and the order Caudovirales. Eleven structural proteins (17 to 200 kDa) for this phage were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A double stranded DNA, approximately 32 kb, in size was detected for this phage on agarose gels. The phage efficacy against E. coli 3 planktonic cells was also investigated. The MJ1 phage demonstrated a very good capability to reduce the numbers of E. coli 3 planktonic cells, as determined by a change in the bacterial growth (an optical density decrease at 600 nm from 0.40 to 0.12). CONCLUSIONS: MJ1 phage has much potential for use in phage therapy and other applications.