Isolation, whole genome sequencing and application of a broad-spectrum Salmonella phage.

Salmonella is considered as one of the most common zoonotic /foodborne pathogens in the world. The application of bacteriophages as novel antibacterial agents in food substrates has become an emerging strategy. Bacteriophages have the potential to control Salmonella contamination.We have isolated and characterized a broad-spectrum Salmonella phage, SP154, which can lyse 9 serotypes, including S. Enteritidis, S. Typhimurium, S. Pullorum, S. Arizonae, S. Dublin, S. Cholerasuis, S. Chester, S. 1, 4, [5], 12: i: -, and S. Derby, accounting for 81.9% of 144 isolates. SP154 showed a short latent period (40 min) and a high burst size (with the first rapid burst size at 107 PFUs/cell and the second rapid burst size at approximately 40 PFUs/cell). Furthermore, SP154 activity has higher survival rates across various environmental conditions, including pH 4.0-12.0 and temperatures ranging from 4 to 50 °C for 60 min, making it suitable for diverse food processing and storage applications. Significant reductions in live Salmonella were observed in different foods matrices such as milk and chicken meat, with a decrease of up to 1.9 log10 CFU/mL in milk contamination and a 1 log10 CFU/mL reduction in chicken meat. Whole genome sequencing analysis revealed that SP154 belongs to the genus Ithacavirus, subfamily Humphriesvirinae, within the family Schitoviridae. Phylogenetic analysis based on the terminase large subunit supported this classification, although an alternate tree using the tail spike protein gene suggested affiliation with the genus Kuttervirus, underscoring the limitations of relying on a single gene for phylogenetic inference. Importantly, no virulence or antibiotic resistance genes were detected in SP154. Our research highlights the potential of using SP154 for biocontrol of Salmonella in the food industry.

Salmonella enteritidis acquires phage resistance through a point mutation in rfbD but loses some of its environmental adaptability.

Phage therapy holds promise as an alternative to antibiotics for combating multidrug-resistant bacteria. However, host bacteria can quickly produce progeny that are resistant to phage infection. In this study, we investigated the mechanisms of bacterial resistance to phage infection. We found that Rsm1, a mutant strain of Salmonella enteritidis (S. enteritidis) sm140, exhibited resistance to phage Psm140, which was originally capable of lysing its host at sm140. Whole genome sequencing analysis revealed a single nucleotide mutation at position 520 (C → T) in the rfbD gene of Rsm1, resulting in broken lipopolysaccharides (LPS), which is caused by the replacement of CAG coding glutamine with a stop codon TAG. The knockout of rfbD in the sm140ΔrfbD strain caused a subsequent loss of sensitivity toward phages. Furthermore, the reintroduction of rfbD in Rsm1 restored phage sensitivity. Moreover, polymerase chain reaction (PCR) amplification of rfbD in 25 resistant strains revealed a high percentage mutation rate of 64% within the rfbD locus. We assessed the fitness of four bacteria strains and found that the acquisition of phage resistance resulted in slower bacterial growth, faster sedimentation velocity, and increased environmental sensitivity (pH, temperature, and antibiotic sensitivity). In short, bacteria mutants lose some of their abilities while gaining resistance to phage infection, which may be a general survival strategy of bacteria against phages. This study is the first to report phage resistance caused by rfbD mutation, providing a new perspective for the research on phage therapy and drug-resistant mechanisms.

Bacteriophage Removal from Infected Salmonella Cultures.

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and interactions. These organisms are viruses that infect and replicate within bacterial hosts. Phages are ubiquitous on Earth, highly diverse, and very abundant. While bacteriophages have valuable roles in different environments and are a key area of research in microbiology and ecology, their presence can be undesirable in certain industrial processes or products. Considering the abundance and ubiquity of bacteriophages on Earth, the design of procedures for the removal of bacteriophages from bacterial cultures is crucial in diverse laboratory and industrial applications to preserve the integrity of the cultures and ensure accurate experimental results or product quality. Here, we have fine-tuned a protocol to eliminate the bacteriophages from infected Salmonella enterica cultures, using a strategy based on the use of lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria. Bacterial LPS plays an important role in host recognition by phages, and we make use of this property to design an effective procedure for the removal of phages, which use LPS as a receptor, in Salmonella bacterial cultures.

Phenotypic characterization and genomic analysis of a Salmonella phage L223 for biocontrol of Salmonella spp. in poultry.

The escalating incidence of foodborne salmonellosis poses a significant global threat to food safety and public health. As antibiotic resistance in Salmonella continues to rise, there is growing interest in bacteriophages as potential alternatives. In this study, we isolated, characterized, and evaluated the biocontrol efficacy of lytic phage L223 in chicken meat. Phage L223 demonstrated robust stability across a broad range of temperatures (20-70 °C) and pH levels (2-11) and exhibited a restricted host range targeting Salmonella spp., notably Salmonella Typhimurium and Salmonella Enteritidis. Characterization of L223 revealed a short latent period of 30 min and a substantial burst size of 515 PFU/cell. Genomic analysis classified L223 within the Caudoviricetes class, Guernseyvirinae subfamily and Jerseyvirus genus, with a dsDNA genome size of 44,321 bp and 47.9% GC content, featuring 72 coding sequences devoid of antimicrobial resistance, virulence factors, toxins, and tRNA genes. Application of L223 significantly (p < 0.005) reduced Salmonella Typhimurium ATCC 14,028 counts by 1.24, 2.17, and 1.55 log CFU/piece after 2, 4, and 6 h of incubation, respectively, in experimentally contaminated chicken breast samples. These findings highlight the potential of Salmonella phage L223 as a promising biocontrol agent for mitigating Salmonella contamination in food products, emphasizing its relevance for enhancing food safety protocols.

Tactical terminase: How a Salmonella prophage navigates oxidative stress.

Stress-induced prophages commonly "jump ship" by inducing lysis via the host SOS response. In a recent work, Uppalapati et al. reports an alternate, stress-selective strategy. Instead of promoting lysis, the Salmonella Gifsy-1 prophage arrests growth specifically when the SOS response coincides with oxidative stress.

Characterization, genome analysis, and therapeutic evaluation of a novel Salmonella phage vB_SalS_JNS02: a candidate bacteriophage for phage therapy.

Phage therapy is gaining momentum as an alternative to antibiotics in the treatment of salmonellosis caused by Salmonella. In this study, a novel Salmonella phage, vB_SalS_JNS02, was isolated successfully from poultry farms in Shandong, China. The biological characteristics of vB_SalS_JNS02 were analysed, which revealed a short latent period of approximately 10 min and a burst size of 110 PFU/cell. Moreover, vB_SalS_JNS02 exhibited remarkable stability across a wide pH range (pH 3-12) and temperatures ranging from 30 to 80°C. Genome sequencing analysis provided valuable insights into the genetic composition of vB_SalS_JNS02, which consists of a double-stranded DNA genome that spans 42,450 base pairs and has a G + C content of 49.4%. Of significant importance, the genomic sequence of vB_SalS_JNS02 did not contain any genes related to lysogenicity, virulence, or antibiotic resistance. The phage's efficacy was evaluated in a larval challenge study. Treatment with the phage resulted in increased survival of Galleria mellonella larvae (100, 70, and 85%) (MOI 0.1) in the prophylactic treatment, co-infection treatment, and remedial treatment experiments, respectively. Another in vivo experiment investigated the potential application of the phage in broiler chickens and revealed that a single oral dose of vB_SalS_JNS02 (108 PFU/mL, 100 µL/chick) administered 3 h after S. enteritidis oral administration provided effective protection. The introduction of bacteriophage not only enhances the production of secretory immunoglobulin A (sIgA), but also induces alterations in the composition of the gut microbial community. Phage therapy increases the relative abundance of beneficial bacteria, which helps to maintain intestinal barrier homeostasis. However, it is unable to fully restore the disrupted intestinal microbiome caused by S. enteritidis infection. Importantly, no significant adverse effects were observed in the animal subjects following oral administration of the phage, and our findings highlight vB_SalS_JNS02 is a hopeful candidate as a promising tool to target Salmonella infections in poultry.

Characterization and efficacy of Salmonella phage cocktail PHA46 in the control of Salmonella Newport and Typhimurium internalized into cherry tomatoes.

Non-typhoid Salmonella enterica causes salmonellosis illness, and this bacterium can contaminate food throughout the production chain, including those that are consumed as raw products. Salmonella enterica can adhere to and internalize into fresh produce such as cherry tomatoes. It has been reported that lytic bacteriophages (phages) can be used as a biocontrol agent in the agricultural field, being an alternative for the control of Salmonella in red meat, fish, lettuce, and cabbage. The aim of this study was to characterize the two phages present in the PHA46 cocktail to determine their morphology, genome, host range, and resistance to different temperatures and pHs values; and later evaluate their lytic activity to reduce the adherence to and internalization of Salmonella enterica serovars Newport and Typhimurium into cherry tomatoes. In addition, in this work, we also explored the effect of the PHA46 cocktail on the virulence of S. Newport-45 and S. Typhimurium SL1344, recovered from the interior of cherry tomatoes, on the lifespan of the animal model Caenorhabditis elegans. The nematode C. elegans, recently has been used to test the virulence of Salmonella and it is easy to maintain and work with in the laboratory. The results revealed that the morphology obtained by Transmission Electron Microscopy of two phages from the PHA46 cocktail correspond to a myovirus, the analyses of their genomes sequences did not report virulence or antimicrobial resistance genes. The PHA46 sample is specific for 33 different serovars from different Salmonella strains and shows stability at 7 °C and pH 6. Also, the PHA46 cocktail was effective in reducing the adherence of S. Newport-45 and S. Typhimurium SL1344 to cherry tomatoes, at an average of 0.9 log10, respectively. Regarding internalized bacteria, the reduction was at an average of 1.2 log10, of the serovars mentioned above. The lifespan experiments in C. elegans showed by itself, that the PHA46 cocktail was harmless to the nematode, and the virulence from both Salmonella strains grown in vitro is diminished in the presence of the PHA46 cocktail. In conclusion, these results showed that the PHA46 cocktail could be a good candidate to be used as a biocontrol agent against Salmonella enterica.

Characterization of vB_SalS_PSa2, a T5-like Demerecviridae bacteriophage, and its potential use in food matrices.

This study characterizes a newly isolated Demerecviridae phage, named vB_SalS_PSa2, belonging to the phage T5 group. The main variations between vB_SalS_PSa2 and T5 concern structural proteins related to morphology and host recognition. vB_SalS_PSa2 is infective to 19 out of the 25 tested Salmonella enterica (including the rare "Sendai" and "Equine" serotypes) and Escherichia coli isolates, most of them being multidrug resistant. vB_SalS_PSa2 displayed good thermal stability (4-60 °C) and broad pH stability (4.0-12.0). It also exhibited antibacterial activity against S. enterica sv. Paratyphi A Enb50 at 4 °C in milk during the whole tested period (5 d), and for 3-6 h at both 25 and 37 °C. Furthermore, vB_SalS_PSa2 was able to inhibit biofilm formation and to show degradation activity on mature biofilms of E. coli K12 and S. enterica sv. Paratyphi Enb50 in both LB and milk. Altogether, these results indicate that phage vB_SalS_PSa2 is a valuable candidate for controlling foodborne S. enterica and E. coli pathogens.

Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5.

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 µg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 µg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 µg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 µg ml-1) and P. aeruginosa P2307 (65.00 µg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

What's on a prophage: analysis of Salmonella spp. prophages identifies a diverse range of cargo with multiple virulence- and metabolism-associated functions.

The gain of mobile elements, such as prophages, can introduce cargo to the recipient bacterium that could facilitate its persistence in or expansion to a new environment, such as a host. While previous studies have focused on identifying and characterizing the genetic diversity of prophages, analyses characterizing the cargo that prophages carry have not been extensively explored. We characterized prophage regions from 303 Salmonella spp. genomes (representing 254 unique serovars) to assess the distribution of prophages in diverse Salmonella. On average, prophages accounted for 3.7% (0.1%-8.8%) of the total genomic content of each isolate. Prophage regions annotated as Gifsy 1 and Salmon Fels 1 were the most commonly identified intact prophages, suggesting that they are common throughout the Salmonella genus. Among 21,687 total coding sequences (CDSs) from intact prophage regions in subsp. enterica genomes, 7.5% (median; range: 1.1%-47.6%) were categorized as having a function not related to prophage integration or phage structure, some of which could potentially provide a functional attribute to the host Salmonella cell. These predicted functions could be broadly categorized into CDSs involved in: (i) modification of cell surface structures (i.e., glycosyltransferases); (ii) modulation of host responses (e.g., SodC/SodA, SopE, ArtAB, and typhoid toxin); (iii) conferring resistance to heavy metals and antimicrobials; (iv) metabolism of carbohydrates, amino acids, and nucleotides; and (v) DNA replication, repair, and regulation. Overall, our systematic analysis of prophage cargo highlights a broader role for prophage cargo in influencing the metabolic, virulence, and resistance characteristics of Salmonella. IMPORTANCE: Lysogenic bacteriophages (phages) can integrate their genome into a bacterial host's genome, potentially introducing genetic elements that can affect the fitness of the host bacterium. The functions of prophage-encoded genes are important to understand as these genes could be mobilized and transferred to a new host. Using a large genomic dataset representing >300 isolates from all known subspecies and species of Salmonella, our study contributes important new findings on the distribution of prophages and the types of cargo that diverse Salmonella prophages carry. We identified a number of coding sequences (CDSs) annotated as having cell surface-modifying attributes, suggesting that prophages may have played an important role in shaping Salmonella's diverse surface antigen repertoire. Furthermore, our characterization of prophages suggests that they play a broader role in facilitating the acquisition and transfer of CDSs associated with metabolism, DNA replication and repair, virulence factors, and to a lesser extent, antimicrobial resistance.

Characterization of two novel Salmonella phages having biocontrol potential against Salmonella spp. in gastrointestinal conditions.

Salmonella is a primary enteric pathogen related to the contamination of poultry and other food products in numerous foodborne outbreaks. The continuous emergence of multidrug-resistant bacteria has become a serious issue due to the overuse of antibiotics. Hence, lytic phages are considered alternative biocontrol agents against these bacterial superbugs. Here, two Salmonella phages-S4lw and D5lw-were subjected to genomic and biological characterization and further encapsulated to improve the stability under acidic conditions mimicking gastrointestinal conditions. The two lytic phages, S4lw and D5lw, taxonomically belong to new species under the Guernseyvirinae and Ackermannviridae families, respectively. Each phage showed antimicrobial activities against diverse Salmonella spp., such as S. Enteritidis and S. Typhimurium, achieving 1.7-3.4 log reduction after 2-6 h of treatment. The phage cocktail at a multiplicity of infection (MOI) of 100 or 1000 completely inhibited these Salmonella strains for at least 14 h at 25 °C. Additionally, the bead-encapsulated phage cocktail could withstand low pH and different simulated gut environments for at least 1 h. Overall, the newly isolated phages can potentially mitigate Salmonella spp. under the gastrointestinal environments through encapsulation and may be further applied via oral administration to resolve common antimicrobial resistance issues in the poultry production chain.

Characterization and resistance mechanism of phage-resistant strains of Salmonella enteritidis.

In the face of the increasingly severe problem of antibiotic resistance, phage therapy is regarded as a highly potential alternative. Compared with traditional antimicrobial agents, a key research area of phage therapy is the study of phage-resistant mutant bacteria. To effectively monitor and prevent this resistance, it is crucial to conduct in-depth exploration of the mechanism behind phage resistance. In this study, a strain of Salmonella enteritidis (sm140) and the corresponding phage (Psm140) were isolated from chicken liver and sewage, respectively. Using the double-layer plate method, successfully screened out phage-resistant mutant strains. Whole-genome resequencing of 3 resistant strains found that the wbaP gene of all 3 strains had mutations at a specific position (1,118), with the base changing from G to A. This mutation causes the gene-encoded glycine to be replaced by aspartic acid. Subsequent studies found that the frequency of this gene mutation is extremely high, reaching 84%, and all mutations occur at the same position. To further explore the relationship between the wbaP gene and phage resistance, knockout strains and complement strains of the wbaP gene were constructed. The experimental results confirmed the association between the wbaP gene and phage resistance. At the same time, biological characteristics and virulence were evaluated for wild strains, resistant strains, knockout strains, and complement strains. It was found that mutations or deletions of the wbaP gene lead to a decrease in bacterial environmental adaptability and virulence. Through systematic research on the mechanism and biological characteristics of phage resistance, this study provides important references and guidance for the development of new phage therapies, promoting progress in the field of antimicrobial treatment. At the same time, the emergence of phage resistance due to wbaP gene mutations is reported for the first time in salmonella, providing a new perspective and ideas for further studying phage resistance mechanisms.

Molecular characterization and environmental impact of newly isolated lytic phage SLAM_phiST1N3 in the Cornellvirus genus for biocontrol of a multidrug-resistant Salmonella Typhimurium in the swine industry chain.

Salmonella Typhimurium is a highly lethal pathogenic bacterium in weaned piglets, causing significant treatment costs and economic losses in the swine industry. Additionally, due to its ability to induce zoonotic diseases, resulting in harm to humans through the transmission of the pathogen from pork, it presents a serious public health issue. Bacteriophages (phages), viruses that infect specific bacterial strains, have been proposed as an alternative to antibiotics for controlling pathogenic bacteria. In this study, we isolated SLAM_phiST1N3, a phage infecting a multidrug-resistant (MDR) S. Typhimurium wild-type strain isolated from diseased pigs. First, comparative genomics and phylogenetic analysis revealed that SLAM_phiST1N3 belongs to the Cornellvirus genus. Moreover, utilizing a novel classification approach introduced in this study, SLAM_phiST1N3 was classified at the species level. Host range experiments demonstrated that SLAM_phiST1N3 did not infect other pathogenic bacteria or probiotics derived from pigs or other livestock. While complete eradication of Salmonella was not achievable in the liquid inhibition assay, surprisingly, we succeeded in largely eliminating Salmonella in the FIMM analysis, a gut simulation system using weaned piglet feces. Furthermore, using the C. elegans model, we showcased the potential of SLAM_phiST1N3 to prevent S. Typhimurium infection in living organisms. In addition, it was confirmed that bacterial control could be achieved when phage was applied to Salmonella-contaminated pork. pH and temperature stability experiments demonstrated that SLAM_phiST1N3 can endure swine industry processes and digestive conditions. In conclusion, SLAM_phiST1N3 demonstrates potential environmental impact as a substance for Salmonella prevention across various aspects of the swine industry chain.

Post-harvest biocontrol of Salmonella Enteritidis on Chicken breast meat and Shell eggs using multiphage cocktail.

The study aimed to evaluate the efficacy of a phage cocktail to reduce Salmonella Enteritidis contamination on perishable food items viz. chicken breast meat and shell eggs using different concentrations. Initially, four bacteriophages €P54, €P59, €P66, and €P72 were isolated from sewage water using Salmonella Enteritidis as a target strain. €P54 and €P66 were found to be Myoviruses while €P59 and €P72 belonged to the Siphoviridae family. A phage cocktail was applied at a concentration of 100 and 10,000 multiplicity of infection (MOI) after artificially contaminating both food items with Salmonella Enteritidis. Results showed that, phage cocktail significantly (p ≤ 0.05) reduced Salmonella Enteritidis count at both concentrations. However, the increased reduction was witnessed at 10,000 MOI. In comparison to untreated control, on chicken breast meat bacterial count was reduced to 1.94 and 3.17 Log10 cfu/g at 100 and 10,000 MOI respectively at 4oC. Similarly, on shell eggs, the bacterial count was reduced to 3.09 and 2.81 Log10 cfu/mL at 10,000 MOI at 4°C and 25°C respectively, while at 100 MOI there was less drop in bacterial count at both 4°C and 25°C. The results showed a better reduction at 4°C as compared to 25°C. Our data showed that the phage cocktail is an effective alternative and additional measure compared to conventional bacterial control methods for meat and eggs.

Genome Analysis and Therapeutic Evaluation of a Novel Lytic Bacteriophage of Salmonella Typhimurium: Suggestive of a New Genus in the Subfamily Vequintavirinae.

Salmonella Typhimurium, a foodborne pathogen, is a major concern for food safety. Its MDR serovars of animal origin pose a serious threat to the human population. Phage therapy can be an alternative for the treatment of such MDR Salmonella serovars. In this study, we report on detailed genome analyses of a novel Salmonella phage (Salmonella-Phage-SSBI34) and evaluate its therapeutic potential. The phage was evaluated for latent time, burst size, host range, and bacterial growth reduction in liquid cultures. The phage stability was examined at various pH levels and temperatures. The genome analysis (141.095 Kb) indicated that its nucleotide sequence is novel, as it exhibited only 1-7% DNA coverage. The phage genome features 44% GC content, and 234 putative open reading frames were predicted. The genome was predicted to encode for 28 structural proteins and 40 enzymes related to nucleotide metabolism, DNA modification, and protein synthesis. Further, the genome features 11 tRNA genes for 10 different amino acids, indicating alternate codon usage, and hosts a unique hydrolase for bacterial lysis. This study provides new insights into the subfamily Vequintavirinae, of which SSBI34 may represent a new genus.

Salmonella Typhimurium DT 104 response to Lytic bacteriophage and Lactobionic acid on raw chicken breast.

Bacterial food poisoning cases due to Salmonella have been linked with a variety of poultry products. This study evaluated the effects of a Salmonella-specific Lytic bacteriophage and Lactobionic acid (LBA) on Salmonella Typhimurium DT 104 growth on raw chicken breast meat. Each chicken breast was randomly assigned to a treatment group (Control, DI water, phage 1%, phage 5%, LBA 10 mg/mL, LBA 20 mg/mL, and phage 5% + LBA 20 mg/mL) with four chicken breasts per group. Samples were inoculated with 106 CFU/mL of Salmonella and stored at 4 °C for 30 min. The inoculated chicken breasts were randomly assigned to different storage time (0 h, 1 h, 24 h, or 48 h). Both time and treatment showed significance reduction (P < 0.0001) of microbial growth. The weight loss was significantly different (P < 0.0001) between treatments. The LBA treatments were not effective when compared to the control group, but Lytic bacteriophage significantly reduced the amount of microbial growth.

Isolation, identification and some characteristics of two lytic bacteriophages against Salmonella enterica serovar Paratyphi B and S. enterica serovar Typhimurium from various food sources.

Salmonellosis is an important worldwide food-borne disease. Increasing resistance to Salmonella spp. has been reported in recent years, and now the prevalence of multidrug-resistant Salmonella spp. is a worldwide problem. This necessitates alternative approaches like phage therapy. This study aimed to isolate bacteriophages specific for Salmonella enterica serovar Paratyphi B and S. enterica serovar Typhimurium isolated from different sources (chicken meat, beef and eggshells). The antibiotic resistance profiles of the bacteria were determined by phenotypic and genotypic methods. The prevalence of extended-spectrum ß-lactamase genes was examined by polymerase chain reaction. In total, 75% of the isolated Salmonella strains were resistant to tetracycline, whereas 70% of them were resistant to azithromycin. All of the isolates from beef were resistant to nalidixic acid. The most common extended-spectrum ß-lactamase genes among the isolates were blaSHV (15%) followed by blaTEM (10%) and blaCTX (5%). Two specific bacteriophages were isolated and characterized. The host range for vB_SparS-ui was Salmonella Paratyphi B, S. enterica serovar Paratyphi A and S. enterica, while that for vB_StyS-sam phage was Salmonella Typhimurium and S. enterica serovar Enteritidis. The characteristics of the isolated phages indicate that they are proper candidates to be used to control some foodstuff contaminations and also phage therapy of infected animals.

Bacteriophage as an alternative to prevent reptile-associated Salmonella transmission.

Salmonellosis is a major global public health issue; its most common infection, gastroenteritis, accounts for approximately 90 million illnesses and 150,000 mortalities per year. Eradicating salmonellosis requires surveillance, prevention and treatment, entailing large expenditures. However, it is difficult to control Salmonella transmission because it occurs via multiple routes; exotic reptiles are a reservoir of Salmonella and comprise one such route. As the popularity of exotic pets and animal exhibition has increased, human encounters with reptiles have also increased. As a result, reptile-associated salmonellosis (RAS) has been recognized as an emerging disease. The development of antimicrobial resistance in RAS-causing Salmonella sp. requires alternatives to antibiotics. In this study, bacteriophages have been established as an alternative to antibiotics because only target bacteria are lysed; thus, they are promising biocontrol agents. Here, bacteriophage pSal-SNUABM-02, which infects and lyses reptile Salmonella isolates, was isolated and characterized. The morphology, host range, growth traits and stability of the phage were investigated. The phage was assigned to Myoviridae and was stable in the following conditions: pH 5-9, 4-37°C, and ultravioletA/ultravioletB (UVA/UVB) exposure. Salmonella clearance efficacy was tested using planktonic cell lysis activity and biofilm degradation on polystyrene 96-well plates and reptile skin fragments. The phage exhibited vigorous lysis activity against planktonic cells. In in vitro biofilm degradation tests on reptile skin and polystyrene plates, both low- and high-concentration phage treatments lowered bacterial cell viability by approximately 2.5-3 log colony-forming units and also decreased biomass. Thus, bacteriophages are a promising alternative to antibiotics for the prevention and eradication of RAS.

Influence of Salmonella specific bacteriophages (O1; S16) on the shedding of naturally occurring Salmonella and an orally applied Salmonella Eastbourne strain in bearded dragons (Pogona vitticeps).

This study determined the passage time and phage propagation time of salmonella specific phages, Felix O1 and S16, in 10 bearded dragons, based on re-isolation from cloacal swabs and faecal samples following oral administration, as a possible tool for reducing salmonella shedding. In Study 1, Felix O1 was administered orally for 12 consecutive days. Over 60 days, swabs were taken from the oral cavity and cloaca and qualitative Salmonella detection as well as salmonella quantification from faecal samples were performed. In Study 2, a phage cocktail (Felix O1 and S16) was administered to half of the tested animals. Salmonella (S.) Eastbourne was also given orally to all animals. Oral and cloacal swabs were tested as in Study 1, and faecal samples were collected for phage quantification. Various Salmonella serovars were detectable at the beginning of the study. The numbers of serovars detected declined over the course of the study. S. Kisarawe was most commonly detected. Salmonella titres ranged from 102 to 107  cfu/g faeces. The phages (Felix O1 and S16) were detectable for up to 20 days after the last administration. The initial phage titres ranged from 103 to 107  pfu/ml. The study shows that the phages were able to replicate in the intestine, and were shed for a prolonged period and therefore could contribute to a reduction of Salmonella shedding.

Isolation and Characterization of Salmonella Jumbo-Phage pSal-SNUABM-04.

The increasing emergence of antimicrobial resistance has become a global issue. Therefore, many researchers have attempted to develop alternative antibiotics. One promising alternative is bacteriophage. In this study, we focused on a jumbo-phage infecting Salmonella isolated from exotic pet markets. Using a Salmonella strain isolated from reptiles as a host, we isolated and characterized the novel jumbo-bacteriophage pSal-SNUABM-04. This phage was investigated in terms of its morphology, host infectivity, growth and lysis kinetics, and genome. The phage was classified as Myoviridae based on its morphological traits and showed a comparatively wide host range. The lysis efficacy test showed that the phage can inhibit bacterial growth in the planktonic state. Genetic analysis revealed that the phage possesses a 239,626-base pair genome with 280 putative open reading frames, 76 of which have a predicted function and 195 of which have none. By genome comparison with other jumbo phages, the phage was designated as a novel member of Machinavirus composed of Erwnina phages.