BtuB-Dependent Infection of the T5-like Yersinia Phage ϕR2-01.

Yersinia enterocolitica is a food-borne Gram-negative pathogen responsible for several gastrointestinal disorders. Host-specific lytic bacteriophages have been increasingly used recently as an alternative or complementary treatment to combat bacterial infections, especially when antibiotics fail. Here, we describe the proteogenomic characterization and host receptor identification of the siphovirus vB_YenS_ϕR2-01 (in short, ϕR2-01) that infects strains of several Yersinia enterocolitica serotypes. The ϕR2-01 genome contains 154 predicted genes, 117 of which encode products that are homologous to those of Escherichia bacteriophage T5. The ϕR2-01 and T5 genomes are largely syntenic, with the major differences residing in areas encoding hypothetical ϕR2-01 proteins. Label-free mass-spectrometry-based proteomics confirmed the expression of 90 of the ϕR2-01 genes, with 88 of these being either phage particle structural or phage-particle-associated proteins. In vitro transposon-based host mutagenesis and ϕR2-01 adsorption experiments identified the outer membrane vitamin B12 receptor BtuB as the host receptor. This study provides a proteogenomic characterization of a T5-type bacteriophage and identifies specific Y. enterocolitica strains sensitive to infection with possible future applications of ϕR2-01 as a food biocontrol or phage therapy agent.

Impact of Phage CDHS-1 on the Transcription, Physiology and Pathogenicity of a Clostridioides difficile Ribotype 027 Strain, R20291.

All known Clostridioides difficile phages encode integrases rendering them potentially able to lyse or lysogenise bacterial strains. Here, we observed the infection of the siphovirus, CDHS-1 on a ribotype 027 strain, R20291 and determined the phage and bacterial gene expression profiles, and impacts of phage infection on bacterial physiology and pathogenicity. Using RNA-seq and RT-qPCR we analysed transcriptomic changes during early, mid-log and late phases of phage replication at an MOI of 10. The phage has a 20 min latent period, takes 80 min to lyse cells and a burst size of ~37. All phage genes are highly expressed during at least one time point. The Cro/C1-transcriptional regulator, ssDNA binding protein and helicase are expressed early, the holin is expressed during the mid-log phase and structural proteins are expressed from mid-log to late phase. Most bacterial genes, particularly the metabolism and toxin production/regulatory genes, were downregulated from early phage replication. Phage-resistant strains and lysogens showed reduced virulence during Galleria mellonella colonization as ascertained by the larval survival and expression of growth (10), reproduction (2) and infection (2) marker genes. These data suggest that phage infection both reduces colonization and negatively impacts bacterial pathogenicity, providing encouraging data to support the development of this phage for therapy to treat C. difficile infection.

Characterization of a Novel Phage ΦAb1656-2 and Its Endolysin with Higher Antimicrobial Activity against Multidrug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial pathogen, which is a problem worldwide due to the emergence of a difficult-to-treat multidrug-resistant A. baumannii (MDRAB). Endolysins are hydrolytic enzymes produced by a bacteriophage that can be used as a potential therapeutic agent for multidrug-resistant bacterial infection in replacing antibiotics. Here, we isolated a novel bacteriophage through prophage induction using mitomycin C from clinical A. baumannii 1656-2. Morphologically, ΦAb1656-2 was identified as a Siphoviridae family bacteriophage, which can infect MDRAB. The whole genome of ΦAb1656-2 was sequenced, and it showed that it is 50.9 kb with a G + C content of 38.6% and 68 putative open reading frames (ORFs). A novel endolysin named AbEndolysin with an N-acetylmuramidase-containing catalytic domain was identified, expressed, and purified from ΦAb1656-2. Recombinant AbEndolysin showed significant antibacterial activity against MDRAB clinical strains without any outer membrane permeabilizer. These results suggest that AbEndolysin could represent a potential antimicrobial agent for treating MDRAB clinical isolates.

Characterization of the first Pseudomonas grimontii bacteriophage, PMBT3.

The complete genome sequence of the virulent bacteriophage PMBT3, isolated on the proteolytic Pseudomonas grimontii strain MBTL2-21, showed no significant similarity to other known phage genome sequences, making this phage the first reported to infect a strain of P. grimontii. Electron microscopy revealed PMBT3 to be a member of the family Siphoviridae, with notably long and flexible whiskers. The linear, double-stranded genome of 87,196 bp has a mol% G+C content of 60.4 and contains 116 predicted protein-encoding genes. A putative tellurite resistance (terB) gene, originally reported to occur in the genome of a bacterium, was detected in the genome of phage PMBT3.

The Bacteriophage pEp_SNUABM_08 Is a Novel Singleton Siphovirus with High Host Specificity for Erwinia pyrifoliae.

Species belonging to the genus Erwinia are predominantly plant pathogens. A number of bacteriophages capable of infecting Erwinia have been used for the control of plant diseases such as fire blight. Public repositories provide the complete genome information for such phages, which includes genomes ranging from 30 kb to 350 kb in size. However, limited information is available regarding bacteriophages belonging to the family Siphoviridae. A novel lytic siphophage, pEp_SNUABM_08, which specifically infects Erwinia pyrifoliae, was isolated from the soil of an affected apple orchard in South Korea. A comprehensive genome analysis was performed using the Erwinia-infecting siphophage. The whole genome of pEp_SNUABM_08 comprised 62,784 bp (GC content, 57.24%) with 79 open reading frames. The genomic characteristics confirmed that pEp_SNUABM_08 is a singleton lytic bacteriophage belonging to the family Siphoviridae, and no closely related phages have been reported thus far. Our study not only characterized a unique phage, but also provides insight into the genetic diversity of Erwinia bacteriophages.

Biogenesis of a Bacteriophage Long Non-Contractile Tail.

Siphoviruses are main killers of bacteria. They use a long non-contractile tail to recognize the host cell and to deliver the genome from the viral capsid to the bacterial cytoplasm. Here, we define the molecular organization of the Bacillus subtilis bacteriophage SPP1 ~ 6.8 MDa tail and uncover its biogenesis mechanisms. A complex between gp21 and the tail distal protein (Dit) gp19.1 is assembled first to build the tail cap (gp19.1-gp21Nter) connected by a flexible hinge to the tail fiber (gp21Cter). The tip of the gp21Cter fiber is loosely associated to gp22. The cap provides a platform where tail tube proteins (TTPs) initiate polymerization around the tape measure protein gp18 (TMP), a reaction dependent on the non-structural tail assembly chaperones gp17.5 and gp17.5* (TACs). Gp17.5 is essential for stability of gp18 in the cell. Helical polymerization stops at a precise tube length followed by binding of proteins gp16.1 (TCP) and gp17 (THJP) to build the tail interface for attachment to the capsid portal system. This finding uncovers the function of the extensively conserved gp16.1-homologs in assembly of long tails. All SPP1 tail components, apart from gp22, share homology to conserved proteins whose coding genes' synteny is broadly maintained in siphoviruses. They conceivably represent the minimal essential protein set necessary to build functional long tails. Proteins homologous to SPP1 tail building blocks feature a variety of add-on modules that diversify extensively the tail core structure, expanding its capability to bind host cells and to deliver the viral genome to the bacterial cytoplasm.

A novel vieuvirus from multidrug-resistant Acinetobacter baumannii.

Bacteriophages are considered the most abundant biological entities on earth, and they are able to modulate the populations of their bacterial hosts. Although the potential of bacteriophages has been accepted as an alternative strategy to combat multidrug-resistant pathogenic bacteria, there still exists a considerable knowledge gap regarding their genetic diversity, which hinders their use as antimicrobial agents. In this study, we undertook a genomic and phylogenetic characterization of the phage Ab11510-phi, which was isolated from a multidrug-resistant Acinetobacter baumannii strain (Ab11510). We found that Ab11510-phi has a narrow host range and belongs to a small group of transposable phages of the genus Vieuvirus that have only been reported to infect Acinetobacter bacteria. Finally, we showed that Ab11510-phi (as well as other vieuvirus phages) has a high level of mosaicism. On a broader level, we demonstrate that comparative genomics and phylogenetic analysis are necessary tools for the proper characterization of phage diversity.

Biological characteristics and genomic analysis of a Stenotrophomonas maltophilia phage vB_SmaS_BUCT548.

Stenotrophomonas maltophilia (hereinafter referred to as S. maltophilia) has developed into an important opportunistic pathogenic bacterium, which is prevalent in nosocomial and community infections, and has adverse effects on patients with a compromised immune system. Phage vB_SmaS_BUCT548 was isolated from sewage of Beijing 307 Hospital with S. maltophilia (strain No.824) as a host. Phage morphology was observed by transmission electron microscopy and its biological and genomic characteristics were determined. The electron microscope shows that the bacteriophage belonged to the Siphoviridae and MOI is 0.001. One-step growth curve shows that the incubation period is 30 min and the burst size is 134 PFU/Cell. The host range is relatively wide and it can lysis 11of 13 S. maltophilia strains. Next-Generation Sequencing (NGS) results show that the genome sequence is a dsDNA with 62354 bp length, and the GC content is 56.3% (GenBank: MN937349). One hundred and two online reading frames (ORFs) are obtained after RAST online annotation and the BlastN nucleic acid comparison shows that the phage had low homology with other phages in NCBI database. This study reports a novel S. maltophilia phage named vB_SmaS_BUCT548, which has a short incubation period, strong lytic ability, and a wide host range. The main characteristic of this bacteriophage is the novelty of the genomic sequence and the analysis of the other characteristics provides basic data for further exploring the interaction mechanism between the phage and the host.

Characterization of bacteriophage VVP001 and its application for the inhibition of Vibrio vulnificus causing seafood-borne diseases.

Vibrio vulnificus is a major food-borne pathogen that causes septicemia and cellulitis with a mortality rate of >50%. However, there are no efficient natural food preservatives or biocontrol agents to control V. vulnificus in seafood. In this study, we isolated and characterized a novel bacteriophage VVP001. Host range and transmission electron microscopy morphology observations revealed that VVP001 belongs to the family Siphoviridae and specifically infects V. vulnificus. Phage stability tests showed that VVP001 is stable at a broad temperature range of -20 °C to 65 °C and a pH range from 3 to 11, which are conditions for food applications (processing, distribution, and storage). In vitro challenge assays revealed that VVP001 inhibited V. vulnificus MO6-24/O (a clinical isolate) growth up to a 3.87 log reduction. In addition, complete genome analysis revealed that the 76 kb VVP001 contains 102 open reading frames with 49.64% G + C content and no gene encoding toxins or other virulence factors, which is essential for food applications. Application of VVP001 to fresh abalone samples contaminated with V. vulnificus demonstrated its ability to inhibit V. vulnificus growth, and an in vivo mouse survival test showed that VVP001 protects mice against high mortality (survival rate >70% at a multiplicity of infection of 1000 for up to 7 days). Therefore, the bacteriophage VVP001 can be used as a good natural food preservative and biocontrol agent for food applications.

Development of a broad-spectrum Salmonella phage cocktail containing Viunalike and Jerseylike viruses isolated from Thailand.

Salmonella is one of the most common agents of foodborne disease worldwide. As natural alternatives to traditional antimicrobial agents, bacteriophages (phages) are emerging as highly effective biocontrol agents against Salmonella and other foodborne bacteria. Due to the high diversity within the Salmonella genus and emergence of drug resistant strains, improved efforts are necessary to find broad range and strictly lytic Salmonella phages for use in food biocontrol. Here, we describe the isolation and characterization of two Salmonella phages: ST-W77 isolated on S. Typhimurium and SE-W109 isolated on S. Enteritidis with extraordinary Salmonella specificity. Whole genome sequencing identified ST-W77 as a Myovirus within the Viunalikevirus genus and SE-W109 as a Siphovirus within the Jerseylikevirus genus. Infectivity studies using a panel of S. Typhimurium cell wall mutants revealed both phages require the lipopolysaccharide O-antigen, with SE-W109 also recognizing the flagella, during infection of Salmonella. A combination of both phages was capable of prolonged (one-week) antibacterial activity when added to milk or chicken meat contaminated with Salmonella. Due to their broad host ranges, strictly lytic lifestyles and lack of lysogeny-related genes or virulence genes in their genomes, ST-W77 and SE-W109 are ideal phages for further development as Salmonella biocontrol agents for food production.

Characterization of φEf-vB1 prophage infecting oral Enterococcus faecalis and enhancing bacterial biofilm formation.

Introduction. Enterococcus faecalis is a facultative, anaerobic, opportunistic pathogen associated with medical and dental diseases. Bacterial phenotypic traits and pathogenesis are often influenced by lysogeny.Aim. The aim of this study was to characterize both the morphology and complete genome sequences of induced prophages purified from E. faecalis clinical isolates.Methodology. E. faecalis isolates were recovered from the roots of teeth of patients attending an endodontic clinic. The morphological features of isolated phage were characterized using transmission electron microscopy (TEM). DNA sequencing was performed using the Illumina MiSeq platform.Results. TEM indicated that the isolated φEf-vB1 prophage belongs to the family Siphoviridae. The φEf-vB1 prophage was stable over a wide range of temperatures and pH. Sequencing of φEf-vB1 DNA revealed that the phage genome is 37 561 bp in length with a G+C content of 37.6mol% and contained 53 ORFs. Comparison with previously predicted prophage genomes using blast revealed that φEf-vB1 has a high sequence similarity to previously characterized phage genomes. The lysogenic E. faecalis strain exhibited a higher biofilm formation capacity relative to the non-lysogenic strain.Conclusion. The current findings highlight the role of lysogeny in modification of E. faecalis properties and reveal the potential importance of prophages in E. faecalis biology and pathogenesis.

Dissolved Microcystin Release Coincident with Lysis of a Bloom Dominated by Microcystis spp. in Western Lake Erie Attributed to a Novel Cyanophage.

Western Lake Erie (Laurentian Great Lakes) is prone to annual cyanobacterial harmful algal blooms (cHABs) dominated by Microcystis spp. that often yield microcystin toxin concentrations exceeding the federal EPA recreational contact advisory of 8 µg liter-1 In August 2014, microcystin levels were detected in finished drinking water above the World Health Organization 1.0 µg liter-1 threshold for consumption, leading to a 2-day disruption in the supply of drinking water for >400,000 residents of Toledo, Ohio (USA). Subsequent metatranscriptomic analysis of the 2014 bloom event provided evidence that release of toxin into the water supply was likely caused by cyanophage lysis that transformed a portion of the intracellular microcystin pool into the dissolved fraction, rendering it more difficult to eliminate during treatment. In August 2019, a similar increase in dissolved microcystins at the Toledo water intake was coincident with a viral lytic event caused by a phage consortium different in composition from what was detected following the 2014 Toledo water crisis. The most abundant viral sequence in metagenomic data sets was a scaffold from a putative member of the Siphoviridae, distinct from the Ma-LMM01-like Myoviridae that are typically documented to occur in western Lake Erie. This study provides further evidence that viral activity in western Lake Erie plays a significant role in transformation of microcystins from the particulate to the dissolved fraction and therefore requires monitoring efforts from local water treatment plants. Additionally, identification of multiple lytic cyanophages will enable the development of a quantitative PCR toolbox to assess viral activity during cHABs.IMPORTANCE Viral attack on cHABs may contribute to changes in community composition during blooms, as well as bloom decline, yet loss of bloom biomass does not eliminate the threat of cHAB toxicity. Rather, it may increase risks to the public by delivering a pool of dissolved toxin directly into water treatment utilities when the dominating Microcystis spp. are capable of producing microcystins. Detecting, characterizing, and quantifying the major cyanophages involved in lytic events will assist water treatment plant operators in making rapid decisions regarding the pool of microcystins entering the plant and the corresponding best practices to neutralize the toxin.

Structure and mechanism of DNA delivery of a gene transfer agent.

Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm.

Identification and Functional Analysis of Temperate Siphoviridae Bacteriophages of Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen that presents a serious clinical challenge due to its increasing resistance to all available antibiotics. Phage therapy has been introduced recently to treat antibiotic-incurable A. baumannii infections. In search for new A. baumannii specific bacteriophages, 20 clinical A. baumannii strains were used in two pools in an attempt to enrich phages from sewage. The enrichment resulted in induction of resident prophage(s) and three temperate bacteriophages, named vB_AbaS_fEg-Aba01, vB_AbaS_fLi-Aba02 and vB_AbaS_fLi-Aba03, all able to infect only one strain (#6597) of the 20 clinical strains, were isolated. Morphological characteristics obtained by transmission electron microscopy together with the genomic information revealed that the phages belong to the family Siphoviridae. The ca. 35 kb genomic sequences of the phages were >99% identical to each other. The linear ds DNA genomes of the phages contained 10 nt cohesive end termini, 52-54 predicted genes, an attP site and one tRNA gene each. A database search revealed an >99% identical prophage in the genome of A.baumannii strain AbPK1 (acc. no. CP024576.1). Over 99% identical prophages were also identified from two of the original 20 clinical strains (#5707 and #5920) and both were shown to be spontaneously inducible, thus very likely being the origins of the isolated phages. The phage vB_AbaS_fEg-Aba01 was also able to lysogenize the susceptible strain #6597 demonstrating that it was fully functional. The phages showed a very narrow host range infecting only two A.baumannii strains. In conclusion, we have isolated and characterized three novel temperate Siphoviridae phages that infect A.baumannii.

Isolation and Characterization of a Lytic Salmonella paratyphi Phage and Its Antibiofilm Activity Individually or Collaborative with Kanamycin Sulfate.

Salmonella is among the most serious of foodborne pathogens worldwide and distributed widely in the natural environment; in addition, it has caused severe medical problems and foodborne diseases. Bacterial biofilm was the multicellular community of microorganisms that attached to nonbiological and biological surfaces. Phages and their derivatives are ideal candidates for replacing and compensating antibiotic resistance problems in the future. In this study, a virulent phage of KM15 was isolated from pig slaughterhouse sump samples in Kunming, China. It belonged to the Siphoviridae family, and optimal growth temperature was 42°C, the pH of optimal preservation buffer was 6-7, optimal multiplicity of infection was 0.0001, and the genome size was 41,869 bp. The Salmonella paratyphi A and Salmonella paratyphi B have a broad spectrum of antibiotic resistance and were isolated from clinical patients in the First People's Hospital of Yunnan Province; fortunately, most of them can be lysed by phage KM15. Collaboration of phage KM15 and kanamycin sulfate has a better antibiofilm effect than KM15 and kanamycin sulfate alone, in low-concentration bacterial culture; KM15 has better antibiofilm effect than kanamycin sulfate in high-concentration bacterial culture. The data of this study provided a strong evidence of application of phage to reduce the growth of Salmonella biofilm, which was important for public health.

Characterization and Genomic Analysis of ValSw3-3, a New Siphoviridae Bacteriophage Infecting Vibrio alginolyticus.

A novel lytic bacteriophage, ValSw3-3, which efficiently infects pathogenic strains of Vibrio alginolyticus, was isolated from sewage water and characterized by microbiological and in silico genomic analyses. Transmission electron microscopy indicated that ValSw3-3 has the morphology of siphoviruses. This phage can infect four species in the Vibrio genus and has a latent period of 15 min and a burst size of 95 ± 2 PFU/infected bacterium. Genome sequencing results show that ValSw3-3 has a 39,846-bp double-stranded DNA genome with a GC content of 43.1%. The similarity between the genome sequences of ValSw3-3 and those of other phages recorded in the GenBank database was below 50% (42%), suggesting that ValSw3-3 significantly differs from previously reported phages at the DNA level. Multiple genome comparisons and phylogenetic analysis based on the major capsid protein revealed that phage ValSw3-3 is grouped in a clade with five other phages, including Listonella phage phiHSIC (GenBank accession no. NC_006953.1), Vibrio phage P23 (MK097141.1), Vibrio phage pYD8-B (NC_021561.1), Vibrio phage 2E1 (KX507045.1), and Vibrio phage 12G5 (HQ632860.1), and is distinct from all known genera within the Siphoviridae family that have been ratified by the International Committee on Taxonomy of Viruses (ICTV). An in silico proteomic comparison of diverse phages from the Siphoviridae family supported this clustering result and suggested that ValSw3-3, phiHSIC, P23, pYD8-B, 2E1, and 12G5 should be classified as a novel genus cluster of Siphoviridae A subsequent analysis of core genes also revealed the common genes shared within this new cluster. Overall, these results provide a characterization of Vibrio phage ValSw3-3 and support our proposal of a new viral genus within the family SiphoviridaeIMPORTANCE Phage therapy has been considered a potential alternative to antibiotic therapy in treating bacterial infections. For controlling the vibriosis-causing pathogen Vibrio alginolyticus, well-documented phage candidates are still lacking. Here, we characterize a novel lytic Vibrio phage, ValSw3-3, based on its morphology, host range and infectivity, growth characteristics, stability under various conditions, and genomic features. Our results show that ValSw3-3 could be a potent candidate for phage therapy to treat V. alginolyticus infections due to its stronger infectivity and better pH and thermal stability than those of previously reported Vibrio phages. Moreover, genome sequence alignments, phylogenetic analysis, in silico proteomic comparison, and core gene analysis all support that this novel phage, ValSw3-3, and five unclassified phages form a clade distant from those of other known genera ratified by the ICTV. Thus, we propose a new viral genus within the Siphoviridae family to accommodate this clade, with ValSw3-3 as a representative member.

Specific Integration of Temperate Phage Decreases the Pathogenicity of Host Bacteria.

Temperate phages are considered as natural vectors for gene transmission among bacteria due to the ability to integrate their genomes into a host chromosome, therefore, affect the fitness and phenotype of host bacteria. Many virulence genes of pathogenic bacteria were identified in temperate phage genomes, supporting the concept that temperate phages play important roles in increasing the bacterial pathogenicity through delivery of the virulence genes. However, little is known about the roles of temperate phages in attenuation of bacterial virulence. Here, we report a novel Bordetella bronchiseptica temperate phage, vB_BbrS_PHB09 (PHB09), which has a 42,129-bp dsDNA genome with a G+C content of 62.8%. Phylogenetic analysis based on large terminase subunit indicated that phage PHB09 represented a new member of the family Siphoviridae. The genome of PHB09 contains genes encoding lysogen-associated proteins, including integrase and cI protein. The integration site of PHB09 is specifically located within a pilin gene of B. bronchiseptica. Importantly, we found that the integration of phage PHB09 significantly decreased the virulence of parental strain B. bronchiseptica Bb01 in mice, most likely through disruption the expression of pilin gene. Moreover, a single shot of the prophage bearing B. bronchiseptica strain completely protected mice against lethal challenge with wild-type virulent B. bronchiseptica, indicating the vaccine potential of lysogenized strain. Our findings not only indicate the complicated roles of temperate phages in bacterial virulence other than simple delivery of virulent genes but also provide a potential strategy for developing bacterial vaccines.

Genomic Characterization, Formulation and Efficacy in Planta of a Siphoviridae and Podoviridae Protection Cocktail against the Bacterial Plant Pathogens Pectobacterium spp.

In the face of global human population increases, there is a need for efficacious integrated pest management strategies to improve agricultural production and increase sustainable food production. To counteract significant food loses in crop production, novel, safe and efficacious measures should be tested against bacterial pathogens. Pectobacteriaceae species are one of the causative agents of the bacterial rot of onions ultimately leading to crop losses due to ineffective control measures against these pathogens. Therefore, the aim of this study was to isolate and characterize bacteriophages which could be formulated in a cocktail and implemented in planta under natural environmental conditions. Transmission electron microscopy (TEM) and genome analysis revealed Siphoviridae and Podoviridae family bacteriophages. To test the protective effect of a formulated phage cocktail against soft rot disease, three years of field trials were performed, using three different methods of treatment application. This is the first study to show the application of a phage cocktail containing Podoviridae and Siphoviridae bacteriophages capable of protecting onions against soft rot in field conditions.

Characterization of Salmonella spp.-specific bacteriophages and their biocontrol application in chicken breast meat.

Chicken breast meat is considered as the main source of Salmonella infection in humans. The aim of this study was to isolate lytic bacteriophages specific for Salmonella enterica serovars Enteritidis and examine their efficacy in a cocktail for the biocontrol of Salmonella spp. in raw chicken breast meat. Four lytic phages belonging to the Myoviridae and Siphoviridae families were isolated from a river proximate to a duck farm. They exhibited broad lytic activities against 11 strains of S. Enteritidis, 11 strains of S. Typhimurium, and one each of S. Paratyphi A, S. San Diego, and S. Typhi. The phages were determined to be stable, exhibited similar degrees of resistance to heat and pH, and had latent periods ranging from 5 to 30 min. In addition, the phage particles were 100% adsorbed within 18 to 40 min. Viable cell counts of bacteria were significantly reduced in raw chicken breast samples (P < 0.05) when treated with a cocktail of all four bacteriophages at 4 °C for 7 days (multiplicities of infection were from 104 to 106 ). These results indicate the potential efficacy of the bacteriophage cocktail as a biological agent against S. Enteritidis in raw chicken breast meat. PRACTICAL APPLICATION: Our findings demonstrate that the phages could be effective in reducing the viability of Salmonella spp. bacteria in chicken breast meat. Therefore, the phage cocktail is a potential bactericidal agent for the biocontrol of Salmonella spp. in raw chicken breast meat and could be used use in various poultry industries in the future.

Characterization of Salmonella bacteriophages and their potential use in dishwashing materials.

AIMS: The aims of this study were to isolate and characterize novel Salmonella phages and to evaluate the effectiveness of phage cocktails used as antibacterial agents in dishwashing materials. METHODS AND RESULTS: The effective phages, vB_STy-RN5i1 and vB_STy-RN29, were isolated from drain water samples collected from open markets using Salmonella Typhimurium as the host strain. These phages were identified as members of Podoviridae and Siphoviridae, respectively. Both phages infected at least six Salmonella serovars and rapidly lysed their host within one hour. They were stable at 4-45°C and at pH 6-9 for at least an hour while being evaluated in this study. The phage application results indicated that bacterial cells were reduced by 3⋅1 and 2⋅7 log CFU per ml at room temperature when they encountered the phage cocktail on scouring pads (SPs) and dishwashing sponges (DSs), respectively. CONCLUSIONS: The isolated Salmonella phages, vB_STy-RN5i1 and vB_STy-RN29, had potential against Salm. Typhimurium and could reduce the occurrence of bacterial-cross-contamination from dishwashing materials, which have been reported to be a source of bacteria, to other kitchen utensils and food. SIGNIFICANCE AND IMPACT OF THE STUDY: The successful reduction of bacterial contamination in dishwashing materials by the phage cocktail consisting of vB_STy-RN5i1 and vB_STy-RN29 reveals its potential to be an alternative antimicrobial agent for SPs and DSs.